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WO2025217610A1 - Systèmes et procédés de synthèse enzymatique d'un oligonucléotide - Google Patents

Systèmes et procédés de synthèse enzymatique d'un oligonucléotide

Info

Publication number
WO2025217610A1
WO2025217610A1 PCT/US2025/024415 US2025024415W WO2025217610A1 WO 2025217610 A1 WO2025217610 A1 WO 2025217610A1 US 2025024415 W US2025024415 W US 2025024415W WO 2025217610 A1 WO2025217610 A1 WO 2025217610A1
Authority
WO
WIPO (PCT)
Prior art keywords
oligonucleotide
reaction chamber
blocking moiety
transferase
aspects
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/024415
Other languages
English (en)
Inventor
David Entwistle
Derek James Clifford GAUNTLETT
Michael Miller
Ryan D. REEVES
David Watts
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Codexis Inc
Original Assignee
Codexis Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Codexis Inc filed Critical Codexis Inc
Publication of WO2025217610A1 publication Critical patent/WO2025217610A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/08—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

  • Described herein are methods for synthesizing oligonucleotides using an enzymatic process. Also described are systems that can be used to perform such methods.
  • Synthetic oligonucleotides are significant reagents in research and medicine and are currently poised to be in greater demand. At present, the pharmaceutical industry is projected to demand hundreds to thousands of kilograms of oligonucleotides per year for active pharmaceutical ingredients (see, e.g., Andrews, Benjamin I et al. “Sustainability Challenges and Opportunities in Oligonucleotide Manufacturing.” The Journal of Organic Chemistry, 2021, 86:49-61). A barrier to widescale and commercial adoption of synthetic oligos as products in multiple fields is the ability to efficiently synthesize RNA, DNA, and other polynucleotides.
  • RNA synthesis using phosphoramidite synthesis chemistry is limited to producing short oligonucleotides of approximately 200 base pairs (Beaucage & Caruthers, Tetrahedron Lett., 1981, 22(20): 1859)
  • optimization of the technology has only been able to achieve incremental gains in oligonucleotide length, yield and efficiency.
  • Phosphoramidite chemistry also requires large amounts of environmentally harmful reagents, such as acetonitrile and dichloroacetic acid, to drive conversion efficiency.
  • environmentally harmful reagents such as acetonitrile and dichloroacetic acid
  • 1 kilogram of small interfering RNA (siRNA) may require as much as 1000 kg of acetonitrile. This results in thousands of kilograms of chemical waste per kilogram of active pharmaceutical ingredient.
  • a method of template-free synthesis of an oligonucleotide comprising: elongating an oligonucleotide in solution by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide using a transferase to make an elongated oligonucleotide comprising the 3' blocking moiety; separating the elongated oligonucleotide comprising the 3' blocking moiety from the transferase; removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the 3' blocking moiety, the elongated oligonucleotide comprising the 3' blocking moiety being in solution when the 3' blocking moiety is removed.
  • the nucleotide comprising the 3' blocking moiety is a preselected type of nucleotide.
  • the 3' blocking moiety is removed from the elongated oligonucleotide comprising the 3' blocking moiety using a hydrolase.
  • the hydrolase is a phosphatase.
  • the hydrolase is an alkaline phosphatase.
  • the method further comprises separating the elongated oligonucleotide without the 3' blocking moiety from the hydrolase.
  • the method further comprises repeating the method for one or more cycles to further elongate the elongated oligonucleotide without the 3' blocking moiety.
  • a plurality of predetermined nucleotides are used to elongate the oligonucleotide, thereby generating an elongated oligonucleotide having a predetermined sequence, wherein each cycle after a first cycle attaches a single nucleotide to a previously elongated oligonucleotide without the 3' blocking moiety.
  • at least two different types of transferases are used in separate cycles.
  • the elongating occurs in a first reaction chamber.
  • the removing the 3' blocking moiety occurs in a second reaction chamber.
  • the oligonucleotide is at least 4 nucleotides in length.
  • the method comprises making the oligonucleotide prior to elongating the oligonucleotide with the transferase.
  • making the oligonucleotide comprises attaching a nucleotide donor to a nucleotide acceptor having a 3'-OH group using a primase.
  • the nucleotide acceptor is a nucleotide monomer.
  • the nucleotide acceptor is a nucleotide polymer.
  • the nucleotide polymer is a 2-mer, 3- mer, 4-mer, 5-mer, 6-mer, or 7-mer.
  • the nucleotide donor is a nucleotide monomer. In some aspects, the nucleotide donor comprises a 3'-blocking moiety. In some aspects, the 3'-blocking moiety of the nucleotide donor is a phosphate. In some aspects, the method comprises removing the 3 '-blocking moiety of the nucleotide donor attached to the nucleotide acceptor to form an elongated nucleotide acceptor. In some aspects, the 3'-blocking moiety is removed from the nucleotide donor using a hydrolase.
  • making the oligonucleotide comprises one or more primase extension cycles, comprising: attaching a nucleotide donor comprising a 3'-blocking moiety to a nucleotide acceptor having a 3'-OH group using a primase to make a 3' -blocked donoracceptor oligonucleotide; inactivating the primase or separating the primase from the 3' -blocked donor-acceptor oligonucleotide; removing the 3'-blocking moiety from the 3' -blocked donor-acceptor oligonucleotide; wherein each cycle uses a new nucleotide donor.
  • the transferase comprises a single strand RNA ligase.
  • the nucleotide comprising the 3' blocking moiety comprises a conjugate moiety, a reactive group, or a linker.
  • the nucleotide comprising the 3' blocking moiety comprises the conjugate moiety, and the conjugate moiety comprises a carbohydrate, a lipid or a lipophilic group, a sterol, a drug, a hormone, a polymer, a protein, a peptide, a toxin, a vitamin, or a combinations thereof.
  • the oligonucleotide comprises a 5' blocking moiety other than a 5'-phosphate moiety or a 5'-OH.
  • the oligonucleotide is elongated under an inert atmosphere.
  • the inert atmosphere is maintained by an inert gas.
  • the inert gas is argon or nitrogen.
  • elongating the oligonucleotide is performed between 35 °C and 45 °C.
  • removing the 3' blocking moiety is performed between 45 °C and 55 °C.
  • the elongated oligonucleotide without the 3' blocking moiety is buffer exchanged and/or concentrated using a desalting chamber.
  • the desalting chamber is maintained between 20 °C and 30 °C.
  • a method of template-free synthesis of an oligonucleotide comprising: in a first reaction chamber, elongating an oligonucleotide in solution by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide using a transferase to make an elongated oligonucleotide comprising the 3' blocking moiety; flowing the elongated oligonucleotide comprising the 3' blocking moiety in the solution to a second reaction chamber, wherein the transferase is retained in the first reaction chamber; in the second reaction chamber, removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the 3' blocking moiety, the elongated oligonucleotide comprising the 3' blocking moiety being in solution when the 3' blocking moiety is removed; and
  • the nucleotide comprising the 3' blocking moiety is a preselected type of nucleotide.
  • the 3' blocking moiety is removed from the elongated oligonucleotide comprising the 3' blocking moiety using a hydrolase.
  • the hydrolase is a phosphatase.
  • the hydrolase is an alkaline phosphatase.
  • the hydrolase is retained in the second reaction chamber when the elongated oligonucleotide without the 3' blocking moiety flows out of the second reaction chamber.
  • the method further comprises repeating the method for one or more cycles to further elongate the elongated oligonucleotide without the 3' blocking moiety.
  • a plurality of predetermined nucleotides are used to elongate the oligonucleotide, thereby generating an elongated oligonucleotide having a predetermined sequence, wherein each cycle after a first cycle attaches a single nucleotide to a previously elongated oligonucleotide without the 3' blocking moiety.
  • at least two different types of transferases are used in separate cycles.
  • the first reaction chamber and the second reaction chamber are connected to each other through one or more conduits, and the elongated oligonucleotide comprising the 3' blocking moiety and the elongated oligonucleotide without the 3' blocking moiety flows through at least a portion of the one or more conduits.
  • the flowing of the elongated oligonucleotide comprising the 3' blocking moiety to the second reaction chamber and/or the flowing of the elongated oligonucleotide without the 3' blocking moiety to the first reaction chamber is controlled by one or more pumps.
  • the flowing of the elongated oligonucleotide comprising the 3' blocking moiety to the second reaction chamber and/or the flowing of the elongated oligonucleotide without the 3' blocking moiety to the first reaction chamber is controlled by one or more valves.
  • the flowing of the elongated oligonucleotide comprising the 3' blocking moiety into the second reaction chamber comprises flowing the elongated oligonucleotide comprising the 3' blocking moiety through an outlet of the first reaction chamber.
  • the flowing of the elongated oligonucleotide comprising the 3' blocking moiety into the second reaction chamber comprises flowing the elongated oligonucleotide comprising the 3' blocking moiety through an inlet of the second reaction chamber. In some aspects, the flowing of the elongated oligonucleotide without the 3' blocking moiety into the first reaction chamber comprises flowing the elongated oligonucleotide comprising the 3' blocking moiety through an outlet of the second reaction chamber.
  • the flowing of the elongated oligonucleotide without the 3' blocking moiety into the first reaction chamber comprises flowing the elongated oligonucleotide comprising the 3' blocking moiety through an inlet of the first reaction chamber.
  • the flowing the elongated oligonucleotide without the 3' blocking moiety from the second reaction chamber into the first reaction chamber comprises: flowing the elongated oligonucleotide without the 3' blocking moiety from the second reaction chamber into one or more reservoirs, and flowing the elongated oligonucleotide without the 3' blocking moiety from the one or more reservoirs into the first reaction chamber.
  • the oligonucleotide is at least 4 nucleotides in length.
  • the method comprises making the oligonucleotide prior to elongating the oligonucleotide with the transferase.
  • making the oligonucleotide comprises attaching a nucleotide donor to a nucleotide acceptor having a 3'-OH group using a primase.
  • the nucleotide acceptor is a nucleotide monomer.
  • the nucleotide acceptor is a nucleotide polymer.
  • the nucleotide polymer is a 2-mer, 3- mer, 4-mer, 5-mer, 6-mer, or 7-mer.
  • the nucleotide donor is a nucleotide monomer.
  • the nucleotide donor comprises a 3'-blocking moiety.
  • the 3'-blocking moiety of the nucleotide donor is a phosphate.
  • the method comprises removing the 3 '-blocking moiety of the nucleotide donor attached to the nucleotide acceptor to form an elongated nucleotide acceptor.
  • the 3'-blocking moiety is removed from the nucleotide donor using a hydrolase.
  • making the oligonucleotide comprises one or more primase extension cycles, comprising: attaching a nucleotide donor comprising a 3'-blocking moiety to a nucleotide acceptor having a 3'-OH group using a primase to make a 3' -blocked donoracceptor oligonucleotide; inactivating the primase or separating the primase from the 3' -blocked donor-acceptor oligonucleotide; removing the 3'-blocking moiety from the 3' -blocked donor-acceptor oligonucleotide; wherein each cycle uses a new nucleotide donor.
  • the transferase comprises a single strand RNA ligase.
  • the nucleotide comprising the 3' blocking moiety comprises a conjugate moiety, a reactive group, or a linker.
  • the nucleotide comprising the 3' blocking moiety comprises the conjugate moiety, and the conjugate moiety comprises a carbohydrate, a lipid or a lipophilic group, a sterol, a drug, a hormone, a polymer, a protein, a peptide, a toxin, a vitamin, or a combinations thereof.
  • the oligonucleotide comprises a 5' blocking moiety other than a 5'-phosphate moiety or a 5'-OH.
  • the oligonucleotide is elongated under an inert atmosphere.
  • the inert atmosphere is maintained by an inert gas.
  • the inert gas is argon or nitrogen.
  • elongating the oligonucleotide is performed between 35 °C and 45 °C.
  • removing the 3' blocking moiety is performed between 45 °C and 55 °C.
  • the elongated oligonucleotide without the 3' blocking moiety is buffer exchanged and/or concentrated using a desalting chamber.
  • the desalting chamber is maintained between 20 °C and 30 °C.
  • a method of template-free synthesis of an oligonucleotide comprising, in a plurality of reaction chambers: in at least a first reaction chamber, elongating an oligonucleotide in solution by attaching a first nucleotide comprising a 3' blocking moiety to the oligonucleotide using a first transferase to make an elongated oligonucleotide comprising the 3' blocking moiety; flowing the elongated oligonucleotide comprising the 3' blocking moiety in the solution from the at least the first reaction chamber into at least a second reaction chamber, wherein the first transferase is retained in the at least the first reaction chamber; in the at least the second reaction chamber, removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the 3' blocking moiety, the elong
  • the first transferase and the second transferase are different types of transferases.
  • the first nucleotide and the second nucleotide are preselected types of nucleotides.
  • the first nucleotide and the second nucleotide are different types of nucleotides.
  • the 3' blocking moiety is removed from the elongated oligonucleotide comprising the 3' blocking moiety using a hydrolase.
  • the hydrolase is a phosphatase.
  • the hydrolase is an alkaline phosphatase.
  • the hydrolase is retained in the at least the second reaction chamber when the elongated oligonucleotide without the 3' blocking moiety flows from the second reaction chamber into the at least the third reaction chamber.
  • the first reaction chamber, the second reaction chamber, and the third reaction chamber are connected through a plurality of conduits, wherein: the elongated oligonucleotide comprising the 3' blocking moiety flows from the at least the first reaction chamber into at least a second reaction chamber through a first portion of the plurality of conduits; and the elongated oligonucleotide without the 3' blocking moiety flows from the at least the second reaction chamber into at least a third reaction chamber through a second portion of the plurality of conduits.
  • the flowing of the elongated oligonucleotide comprising the 3' blocking moiety into the at least the second reaction chamber and/or the flowing of the elongated oligonucleotide without the 3' blocking moiety into the at least the third reaction chamber is controlled by one or more pumps. In some aspects, the flowing of the elongated oligonucleotide comprising the 3' blocking moiety into the at least the second reaction chamber and/or the flowing of the elongated oligonucleotide without the 3' blocking moiety into the at least the third reaction chamber is controlled by one or more valves.
  • the nucleotide comprising a 3' blocking moiety is a nucleotide triphosphate comprising a 3' blocking moiety or an analog thereof comprising a 5' phosphate analog.
  • the 5' phosphate analog is a 5'-(a- P-thio)phosphate moiety.
  • the hydrolase removes the 3' blocking moiety from unreacted nucleotides in the solution. In some aspects, the hydrolase removes one or more 5' phosphate moieties and/or 5' phosphate analogs from unreacted nucleotides in the solution.
  • the hydrolase removes three 5' phosphate moieties and/or 5' phosphate analogs from unreacted nucleotides in the solution.
  • the hydrolase is immobilized on a solid support.
  • the hydrolase is immobilized using a covalent, electrostatic, or ionic bond.
  • the transferase is a polymerase from the DNA polymerase X family.
  • the transferase is a template independent transferase.
  • the transferase is a terminal deoxynucleotidyl transferase (TdT).
  • TdT terminal deoxynucleotidyl transferase
  • the transferase is immobilized on a solid support.
  • the transferase is immobilized using a covalent, electrostatic, or ionic bond.
  • elongating produces an inorganic pyrophosphate byproduct.
  • the method further comprises degrading the inorganic pyrophosphate using a pyrophosphatase.
  • the pyrophosphatase is an inorganic pyrophosphatase.
  • the pyrophosphatase is immobilized on a solid support.
  • the pyrophosphatase is immobilized using a covalent, electrostatic, or ionic bond.
  • the elongating and the degrading occur within the same reaction chamber.
  • the transferase and the inorganic pyrophosphatase are fused together. In some aspects, the transferase and the pyrophosphatase are immobilized on the same solid support. In some aspects, the transferase and the pyrophosphatase are immobilized on different solid supports. In some aspects, the transferase is immobilized on a solid support and the pyrophosphatase is in the solution. In some aspects, the pyrophosphatase is retained in the reaction chamber comprising the transferase, wherein the reaction chamber comprises a filter that prevents passage of the pyrophosphatase and allows passage of the oligonucleotide.
  • the elongating and the degrading occur within different reaction chambers.
  • the method comprises flowing the elongated oligonucleotide comprising the 3' blocking moiety and the inorganic pyrophosphate byproduct, in the solution, from a reaction chamber comprising the transferase into a reaction chamber comprising the pyrophosphatase, followed by flowing the elongated oligonucleotide comprising the 3' blocking moiety from the reaction chamber comprising the pyrophosphatase into a reaction chamber in which the 3' blocking moiety is removed from the elongated oligonucleotide comprising the 3' blocking moiety.
  • the first reaction chamber or the second reaction chamber is a column.
  • the first reaction chamber comprises a fixed bed comprising the transferase immobilized on a solid support. In some aspects, the first reaction chamber comprises a fluidized bed comprising the transferase immobilized on a solid support. In some aspects, the first reaction chamber comprises a filter that prevents passage of the transferase and allow s passage of the oligonucleotide. In some aspects, the second reaction chamber comprises a fixed bed comprising the hydrolase immobilized on a solid support. In some aspects, the second reaction chamber comprises a fluidized bed comprising the hydrolase immobilized on a solid support. In some aspects, the second reaction chamber comprises a filter that prevents passage of hydrolase and allows passage of the oligonucleotide.
  • the first reaction chamber or the second reaction chamber is a batch reaction chamber.
  • the batch reaction chamber comprises an impeller.
  • the first reaction chamber comprises a rotating bed reactor comprising the transferase immobilized on a solid support.
  • the second reaction chamber comprises a rotating bed reactor comprising hydrolase immobilized on a solid support.
  • the method further comprises separating unreacted nucleotides or reaction byproducts in the solution from the oligonucleotide.
  • the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using liquid chromatography.
  • the liquid chromatography comprises size exclusion chromatography.
  • the liquid chromatography comprises reverse phase chromatography. In some aspects, the liquid chromatography comprises ion exchange chromatography. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using dialysis. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using tangential flow filtration. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide after elongating the oligonucleotide by four or more nucleotides.
  • the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide only after elongating the oligonucleotide by four or more nucleotides.
  • the 3' blocking moiety is a phosphate moiety.
  • the nucleotide comprising the 3' blocking moiety is a ribonucleotide.
  • the nucleotide comprising the 3' blocking moiety is a deoxyribonucleotide.
  • the nucleotide comprising the 3' blocking moiety comprises a 2' modification.
  • the 2' modification is 2'-F or 2'-0-Me (2’-O-methyl).
  • the nucleotide comprising the 3' blocking moiety comprises a nucleoside 5'-(a-P-thio)phosphate.
  • the oligonucleotide comprises a 5' modification.
  • the oligonucleotide is at least 4 nucleotides in length.
  • the method comprises making the oligonucleotide prior to elongating the oligonucleotide with the transferase.
  • making the oligonucleotide comprises attaching a nucleotide donor to a nucleotide acceptor having a 3'-OH group using a primase.
  • the nucleotide acceptor is a nucleotide monomer. In some aspects, the nucleotide acceptor is a nucleotide polymer. In some aspects, the nucleotide polymer is a 2-mer, 3-mer, 4-mer, 5-mer, 6-mer, or 7-mer. In some aspects, the nucleotide donor is a nucleotide monomer. In some aspects, the nucleotide donor comprises a 3'-blocking moiety. In some aspects, the 3'-blocking moiety of the nucleotide donor is a phosphate.
  • the method comprises removing the 3'-blocking moiety of the nucleotide donor attached to the nucleotide acceptor to form an elongated nucleotide acceptor.
  • the 3'- blocking moiety is removed from the nucleotide donor using a hydrolase.
  • making the oligonucleotide comprises one or more primase extension cycles, comprising: attaching a nucleotide donor comprising a 3'-blocking moiety to a nucleotide acceptor having a 3'-OH group using a primase to make a 3' -blocked donor-acceptor oligonucleotide; inactivating the primase or separating the primase from the 3' -blocked donor-acceptor oligonucleotide; removing the 3'-blocking moiety from the 3' -blocked donor-acceptor oligonucleotide; wherein each cycle uses a new nucleotide donor.
  • the transferase comprises a single strand RNA ligase.
  • the nucleotide comprising the 3' blocking moiety comprises a conjugate moiety, a reactive group, or a linker.
  • the nucleotide comprising the 3' blocking moiety comprises the conjugate moiety, and the conjugate moiety comprises a carbohydrate, a lipid or a lipophilic group, a sterol, a drug, a hormone, a polymer, a protein, a peptide, a toxin, a vitamin, or a combinations thereof.
  • the oligonucleotide comprises a 5' blocking moiety other than a 5'-phosphate moiety or a 5'- OH.
  • the oligonucleotide is elongated under an inert atmosphere.
  • the inert atmosphere is maintained by an inert gas.
  • the inert gas is argon or nitrogen.
  • elongating the oligonucleotide is performed between 35 °C and 45 °C.
  • removing the 3' blocking moiety is performed between 45 °C and 55 °C.
  • the elongated oligonucleotide without the 3' blocking moiety is buffer exchanged and/or concentrated using a desalting chamber.
  • the desalting chamber is maintained between 20 °C and 30 °C.
  • a system for template-free synthesis of an oligonucleotide comprising: a first reaction chamber comprising a transferase; and a second reaction chamber comprising a hydrolase; wherein the system is configured to flow an oligonucleotide in a solution from the first reaction chamber to the second reaction chamber while retaining the transferase in the first reaction chamber, and flow said oligonucleotide in the solution from the second chamber back to the first reaction chamber while retaining the hydrolase in the second reaction chamber.
  • the system further comprises a temperature regulator that controls a temperature of the solution in the system.
  • the temperature regulator is configured to control the temperature of the solution in the first reaction chamber or the second reaction chamber.
  • the system further comprises one or more conduits that connects the first reaction chamber and the second reaction chamber.
  • the temperature regulator is a jacketed stir tank.
  • the system further comprises an in-line temperature regulator that controls a temperature of the solution in at least one of the one or more conduits.
  • the one or more conduits comprises a first set of conduits connecting an outlet of the first reaction chamber to an inlet of the second reaction chamber, and a second set of conduits connecting an outlet of the second reaction chamber to an inlet of the first reaction chamber.
  • the system comprises one or more pumps configured to flow the solution from the first reaction chamber to the second reaction chamber, and from the second reaction chamber to the first reaction chamber.
  • the one or more pumps are configured to control a flow rate of the solution.
  • the system further comprises a third reaction chamber comprising a second transferase, wherein the system is further configured to selectively flow said oligonucleotide in the solution from the second chamber to the third reaction chamber while retaining the hydrolase in the second reaction chamber, and flow the oligonucleotide in the solution from the third reaction chamber to the second reaction chamber while retaining the second transferase in the third reaction chamber.
  • the transferase and the second transferase are different types of transferase.
  • the system further comprises one or more valves that selectively controls a flow pathway of the solution in the system.
  • the hydrolase can remove a 3' blocking moiety from the oligonucleotide in the solution.
  • the hydrolase is a phosphatase.
  • the hydrolase is an alkaline phosphatase.
  • transferase can react a nucleotide triphosphate (NTP) comprising a 3' blocking moiety, or an analog thereof comprising a 5' phosphate analog, with the oligonucleotide to elongate the oligonucleotide.
  • NTP nucleotide triphosphate
  • the 5' phosphate analog is a 5'-(a-P-thio)phosphate moiety.
  • the NTP comprising the 3' blocking moiety or the analog thereof is a ribonucleotide. In some aspects, the NTP comprising the 3' blocking moiety or the analog thereof is a deoxyribonucleotide. In some aspects, the NTP comprising the 3' blocking moiety or the analog thereof comprises a 2' modification. In some aspects, the 2' modification of the NTP or the analog thereof is 2'-F or 2'-0Me. In some aspects, the 3' blocking moiety of the NTP or the analog thereof is a phosphate moiety. In some aspects, the hydrolase can remove a 3' blocking moiety from unreacted nucleotides comprising a 3' blocking moiety in the solution.
  • the unreacted nucleotides are ribonucleotides. In some aspects, the unreacted nucleotides comprise a 2' modification. In some aspects, the 2' modification of the unreacted nucleotides is 2'-F or 2'-0Me. In some aspects, the 3' blocking moiety of the unreacted nucleotides is a phosphate moiety. In some aspects, the unreacted nucleotides comprise a 5' phosphate analog. In some aspects, the 5' phosphate analog of the unreacted nucleotides is a 5'-(a-P-thio)phosphate moiety.
  • the hydrolase can remove one or more 5' phosphate moieties and/or 5'-(a-P-thio)phosphate moieties from the unreacted nucleotides in the solution.
  • the hydrolase is immobilized on a solid support.
  • the hydrolase is immobilized using a covalent, electrostatic, or ionic bond.
  • the transferase is a polymerase from the DNA polymerase X family.
  • the transferase is a template independent transferase.
  • the transferase is a terminal deoxynucleotidyl transferase (TdT).
  • TdT terminal deoxynucleotidyl transferase
  • the transferase is immobilized on a solid support.
  • the transferase is immobilized using a covalent, electrostatic, or ionic bond.
  • the first reaction chamber further comprises a pyrophosphatase.
  • the system further comprises a third reaction chamber comprising a pyrophosphatase, wherein the system is configured to flow the oligonucleotide in the solution from the first reaction chamber to the third reaction chamber comprising the pyrophosphatase while retaining the transferase in the first reaction chamber, and selectively flow said oligonucleotide in the solution from the third reaction chamber comprising the pyrophosphatase to the first reaction chamber comprising the transferase or the second reaction chamber comprising the hydrolase, wherein the pyrophosphate is retained in the third reaction chamber comprising the pyrophosphatase.
  • the pyrophosphatase is an inorganic pyrophosphatase. In some aspects, the pyrophosphatase is immobilized on a solid support. In some aspects, the pyrophosphatase is immobilized using a covalent, electrostatic, or ionic bond. In some aspects, the transferase and phosphatase are fused together. In some aspects, the transferase and phosphatase are immobilized on the same solid support. In some aspects, the first reaction chamber or the second reaction chamber is a column. In some aspects, the first reaction chamber comprises a fixed bed comprising the transferase immobilized on a solid support.
  • the first reaction chamber comprises a fluidized bed comprising the transferase immobilized on a solid support. In some aspects, the first reaction chamber comprises a filter that prevents passage of the transferase and allows passage of the oligonucleotide. In some aspects, the second reaction chamber comprises a fixed bed comprising the hydrolase immobilized on a solid support. In some aspects, the second reaction chamber comprises a fluidized bed comprising the hydrolase immobilized on a solid support. In some aspects, the second reaction chamber comprises a filter that prevents passage of the hydrolase and allows passage of the oligonucleotide. In some aspects, the first reaction chamber or the second reaction chamber is a batch reaction chamber. In some aspects, the batch reaction chamber comprises an impeller.
  • the first reaction chamber comprises a rotating bed reactor comprising the transferase immobilized on a solid support.
  • the second reaction chamber comprises a rotating bed reactor comprising the hydrolase immobilized on a solid support.
  • the system further comprises a purification chamber configured to separate unreacted nucleotides or reaction byproducts in the solution from an oligonucleotide.
  • the purification chamber comprises a column.
  • the purification chamber comprises a liquid chromatography column.
  • the purification chamber is a size exclusion column.
  • the purification chamber is an ion exchange column.
  • the purification chamber is reverse phase column.
  • purification chamber is part of a tangential flow filtration system.
  • the first reaction chamber or the second reaction chamber are cleanable or replaceable.
  • the first reaction chamber comprises an inlet and an outlet;
  • the second reaction chamber comprises an inlet and an outlet; and the system further comprising a reagent reservoir comprising an inlet and an outlet; and wherein the system is configured to flow the oligonucleotide in the solution from an outlet of one of the reagent reservoir to the inlet of the first reaction chamber, from the outlet of the first reaction chamber to the inlet of the second reaction chamber, and from the outlet of the second reaction chamber to an inlet of the reagent reservoir.
  • the reagent reservoir comprises an impeller.
  • the reagent reservoir is replaceable or cleanable.
  • the method further comprises a product reservoir comprising an inlet and an outlet, wherein the system is configured to flow the oligonucleotide in the solution from an outlet of the second reaction chamber to an inlet of the product reservoir, from an outlet of the product reservoir to an inlet of the reagent reservoir.
  • the system further comprises a product reservoir comprising an inlet and an outlet, wherein the system is configured to flow the oligonucleotide in the solution from an outlet of the second reaction chamber to an inlet of the product reservoir, from an outlet of the product reservoir to an inlet of the first reaction chamber.
  • the product reservoir comprises an impeller.
  • the product reservoir is replaceable or cleanable.
  • the reagent reservoir comprises the oligonucleotide and a nucleotide comprising a 3' blocking moiety.
  • the system further comprises the purification chamber, wherein the purification chamber comprises an inlet and an outlet, wherein the system is configured to flow the oligonucleotide in the solution, in order, from the outlet of the second reaction chamber to the inlet of the purification chamber, and from the outlet of the purification chamber to the inlet of the reagent reservoir.
  • the system comprises the purification chamber, wherein the system is configured to flow the oligonucleotide, in the solution, from the outlet of the reagent reservoir to the inlet of the first reaction chamber, from the outlet of the first reaction chamber to the inlet of the second reaction chamber, from the outlet of the second reaction chamber to the inlet of the purification chamber, from the outlet of the purification chamber to the inlet of the product reservoir, and from the outlet of the product reservoir to the inlet of the reagent reservoir.
  • the first reaction comprises an inlet and an outlet;
  • the second reaction chamber comprises an inlet and an outlet; and the system further comprising a reagent reservoir comprising an inlet and an outlet, and one or more diverter valves configured to control a flow pathway of the solution; wherein the system is configured to controllably flow the oligonucleotide in the solution through the flow pathway selected from a plurality of flow pathways comprising (i) a first flow pathway comprising flow of the oligonucleotide in the solution from the outlet of the reagent reservoir to the inlet of the first reaction chamber without flowing through the second reaction chamber, and (ii) a second flow pathway comprising flow of the oligonucleotide in the solution from the outlet of the reagent reservoir to the inlet of the second reaction chamber without flowing through the first reaction chamber.
  • the system is configured to automatically select the flow pathway.
  • the first flow pathway comprises flow of the solution from the outlet of the first reaction chamber to the inlet of the reagent reservoir without flowing through the second reaction chamber; and the second flow pathway comprises flow of the solution from the outlet of the second reaction chamber to the inlet of the reagent reservoir without flowing through the first reaction chamber.
  • the system further comprises the purification chamber comprising an inlet and an outlet, and the plurality of flow pathways further comprises a third flow pathway comprising flow of the oligonucleotide in solution from the outlet of the reagent reservoir to the inlet of the purification chamber without flowing through the first reaction chamber or the second reaction chamber.
  • the third flow pathway further comprises flow of the solution from the outlet of the purification chamber to the inlet of the reagent reservoir without flowing through the first reaction chamber or the second reaction chamber.
  • the system further comprises the purification chamber comprising an inlet and an outlet, and the plurality of flow pathways further comprises a fourth flow pathway comprising flow of the oligonucleotide in solution from the outlet of the reagent chamber to an inlet of the first reaction chamber, from an outlet of the first reaction chamber into an inlet of the purification chamber, and from an outlet of the purification chamber into an inlet of the reagent reservoir.
  • the system further comprises the purification chamber comprising an inlet and an outlet
  • the plurality of flow pathways further comprises a fifth flow pathway comprising flow of the oligonucleotide in solution from the outlet of the reagent chamber to an inlet of the second reaction chamber, from an outlet of the second reaction chamber into an inlet of the purification chamber, and from an outlet of the purification chamber into an inlet of the reagent reservoir.
  • the reagent reservoir comprises an impeller.
  • the transferase in the first reaction chamber is immobilized on a solid support, and the first reaction chamber comprises an inlet, an outlet, and an impeller configured to suspend the solid support comprising the transferase; and the hydrolase in the second reaction chamber is immobilized on a solid support, and the second reaction chamber comprises an inlet, an outlet, and an impeller configured to suspend the solid support comprising the hydrolase.
  • the transferase in the first reaction chamber is immobilized on a solid support within a rotating bed reactor, and the first reaction chamber comprises an inlet and an outlet; and the hydrolase in the second reaction chamber is immobilized on a solid support within a rotating bed reactor, and the second reaction chamber comprises an inlet and an outlet.
  • the system comprises one or more diverter valves configured to alternatively direct flow of the solution through (i) a first flow pathways comprising flow of the solution from the outlet of the first reaction chamber to an inlet of the purification chamber, and from an outlet of the purification chamber to the inlet of the first reaction chamber, or (ii) a second flow pathway comprising flow of the solution from the outlet of the first reaction chamber to the inlet of the second reaction chamber, and from an outlet of the second reaction chamber to the inlet of the first reaction chamber.
  • the system further comprises a reaction chamber comprising a primase. In some aspects, the primase is immobilized on a solid support.
  • the reaction chamber comprising the primase comprises a filter that prevents passage of the primase and allows passage of a 3' -blocked donor-acceptor oligonucleotide.
  • the system comprises a degassing system or a sparging system.
  • the system comprises a degassing system, and the degassing system comprises a vacuum pump.
  • the system comprises the sparging system.
  • the sparging system is an in-line sparging system or is configured to sparge liquids in a reservoir.
  • the unit for sparging system is configured to sparge using an inert gas.
  • the inert gas is argon or nitrogen.
  • the system comprises a degasser configured to remove oxygen from the system.
  • the purification chamber is configured to concentrate the elongated oligonucleotide comprising the 3' blocking moiety.
  • the purification chamber comprises a membrane with a molecular weight cutoff of about 500 kDa to about 5000 kDa.
  • a method of template-free synthesis of an oligonucleotide comprising: flowing an oligonucleotide, in a solution, from a reagent reservoir into a first reaction chamber comprising a transferase; in the first reaction chamber, elongating the oligonucleotide by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide using the transferase to make an elongated oligonucleotide comprising the 3' blocking moiety; flowing the elongated oligonucleotide comprising the nucleotide comprising the 3' blocking moiety in the solution to a second reaction chamber comprising a hydrolase, wherein the transferase is retained in the first reaction chamber; in the second reaction chamber, removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety in the solution to make an elongated
  • the method further comprises mixing the oligonucleotide and the nucleotides comprising a 3'-blocking moiety in the reservoir. In some aspects, the method further comprises flowing the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety, in the solution, through a purification chamber configured to separate unreacted nucleotides or reaction byproducts in the solution from the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety before flowing the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety into the reagent reservoir.
  • the method further comprises flowing the oligonucleotide without the 3' blocking moiety into a product reservoir before flowing the oligonucleotide without the 3' blocking moiety into the reagent reservoir. In some aspects, the method further comprises replacing or cleaning the reagent reservoir before flowing the elongated oligonucleotide without the 3' blocking moiety into the reagent reservoir. In some aspects, the method repeats the method for at least two cycles to further elongate the elongated oligonucleotide without the 3' blocking moiety. In some aspects, the method further comprising flowing buffer without the oligonucleotide through the system between cycles.
  • the oligonucleotide is at least 4 nucleotides in length.
  • the method comprises making the oligonucleotide prior to elongating the oligonucleotide with the transferase.
  • making the oligonucleotide comprises attaching a nucleotide donor to a nucleotide acceptor having a 3'-OH group using a primase.
  • the nucleotide acceptor is a nucleotide monomer.
  • the nucleotide acceptor is a nucleotide polymer.
  • the nucleotide polymer is a 2-mer, 3-mer, 4-mer, 5-mer, 6-mer, or 7-mer.
  • the nucleotide donor is a nucleotide monomer.
  • the nucleotide donor comprises a 3'-blocking moiety.
  • the 3'-blocking moiety of the nucleotide donor is a phosphate.
  • the method comprises removing the 3'-blocking moiety of the nucleotide donor attached to the nucleotide acceptor to form an elongated nucleotide acceptor.
  • the 3'- blocking moiety is removed from the nucleotide donor using a hydrolase.
  • making the oligonucleotide comprises one or more primase extension cycles, comprising: attaching a nucleotide donor comprising a 3'-blocking moiety to a nucleotide acceptor having a 3'-OH group using a primase to make a 3' -blocked donor-acceptor oligonucleotide; inactivating the primase or separating the primase from the 3' -blocked donor-acceptor oligonucleotide; removing the 3'-blocking moiety from the 3' -blocked donor-acceptor oligonucleotide; wherein each cycle uses a new nucleotide donor.
  • the transferase comprises a single strand RNA ligase.
  • the nucleotide comprising the 3' blocking moiety comprises a conjugate moiety, a reactive group, or a linker.
  • the nucleotide comprising the 3' blocking moiety comprises the conjugate moiety, and the conjugate moiety comprises a carbohydrate, a lipid or a lipophilic group, a sterol, a drug, a hormone, a polymer, a protein, a peptide, a toxin, a vitamin, or a combinations thereof.
  • the oligonucleotide comprises a 5' blocking moiety other than a 5'-phosphate moiety or a 5'- OH.
  • the oligonucleotide is elongated under an inert atmosphere.
  • the inert atmosphere is maintained by an inert gas.
  • the inert gas is argon or nitrogen.
  • elongating the oligonucleotide is performed between 35 °C and 45 °C.
  • removing the 3' blocking moiety is performed between 45 °C and 55 °C.
  • the elongated oligonucleotide without the 3' blocking moiety is buffer exchanged and/or concentrated using a desalting chamber.
  • the desalting chamber is maintained between 20 °C and 30 °C.
  • a method of template-free synthesis of an oligonucleotide comprising: (a) elongating an oligonucleotide in a solution by attaching a nucleotide comprising a 3' blocking moiety to an oligonucleotide transferase to make an elongated oligonucleotide comprising the 3' blocking moiety, the elongating comprising subjecting the oligonucleotide to one or more elongation flow pathway cycles comprising: flowing the oligonucleotide and nucleotides comprising a 3' blocking moiety from a reagent reservoir to a first reaction chamber comprising a transferase, and flowing the oligonucleotide from the first reaction chamber to the reservoir; and (b) removing the 3' blocking moiety from the oligonucleotide in the solution to make an elongated oligonucleotide without the 3' blocking moiety
  • the oligonucleotide is at least 4 nucleotides in length.
  • the method comprises making the oligonucleotide prior to elongating the oligonucleotide with the transferase.
  • making the oligonucleotide comprises attaching a nucleotide donor to a nucleotide acceptor having a 3'-OH group using a primase.
  • the nucleotide acceptor is a nucleotide monomer.
  • the nucleotide acceptor is a nucleotide polymer.
  • the nucleotide polymer is a 2-mer, 3-mer, 4-mer, 5-mer, 6-mer, or 7-mer.
  • the nucleotide donor is a nucleotide monomer.
  • the nucleotide donor comprises a 3'-blocking moiety.
  • the 3'-blocking moiety of the nucleotide donor is a phosphate.
  • the method comprises removing the 3 '-blocking moiety of the nucleotide donor attached to the nucleotide acceptor to form an elongated nucleotide acceptor.
  • the 3'-blocking moiety is removed from the nucleotide donor using a hydrolase.
  • making the oligonucleotide comprises one or more primase extension cycles, comprising: attaching a nucleotide donor comprising a 3'-blocking moiety to a nucleotide acceptor having a 3'-OH group using a primase to make a 3' -blocked donoracceptor oligonucleotide; inactivating the primase or separating the primase from the 3' -blocked donor-acceptor oligonucleotide; removing the 3'-blocking moiety from the 3' -blocked donor-acceptor oligonucleotide; wherein each cycle uses a new nucleotide donor.
  • the transferase comprises a single strand RNA ligase.
  • the nucleotide comprising the 3' blocking moiety comprises a conjugate moiety, a reactive group, or a linker.
  • the nucleotide comprising the 3' blocking moiety comprises the conjugate moiety, and the conjugate moiety comprises a carbohydrate, a lipid or a lipophilic group, a sterol, a drug, a hormone, a polymer, a protein, a peptide, a toxin, a vitamin, or a combinations thereof.
  • the oligonucleotide comprises a 5' blocking moiety other than a 5'-phosphate moiety or a 5'-OH.
  • the oligonucleotide is elongated under an inert atmosphere.
  • the inert atmosphere is maintained by an inert gas.
  • the inert gas is argon or nitrogen.
  • elongating the oligonucleotide is performed between 35 °C and 45 °C.
  • removing the 3' blocking moiety is performed between 45 °C and 55 °C.
  • the elongated oligonucleotide without the 3' blocking moiety is buffer exchanged and/or concentrated using a desalting chamber.
  • the desalting chamber is maintained between 20 °C and 30 °C.
  • a method of template-free synthesis of an oligonucleotide comprising: (a) elongating an oligonucleotide in a solution by attaching a nucleotide comprising a 3' blocking moiety to an oligonucleotide transferase to make an elongated oligonucleotide comprising the 3' blocking moiety, the elongating comprising subjecting the oligonucleotide to one or more elongation flow pathway cycles comprising: flowing the oligonucleotide and 3'-blocked nucleotides from a reagent reservoir to a first reaction chamber comprising a transferase, and flowing the oligonucleotide from the first reaction chamber to the reservoir; (b) flowing the elongated oligonucleotide comprising the 3' blocking moiety from the reagent reservoir to a second reaction chamber comprising a hydrolase; and (c) removing the
  • the elongating comprises subjecting the oligonucleotide to a plurality of elongation flow path cycles.
  • the removing comprises subjecting the oligonucleotide to a plurality of deblocking flow pathway cycles.
  • the method comprises separating unreacted nucleotides or reaction byproducts in the solution from the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety.
  • the separating comprises subjecting the oligonucleotide to one or more purification flow pathway cycles comprising: flowing the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety from the reagent reservoir or the product reservoir to a purification chamber configured to separate the unreacted nucleotides or reaction byproducts from the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety, and flowing the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety from the purification chamber to the reagent reservoir or the product reservoir.
  • the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using liquid chromatography.
  • the liquid chromatography comprises size exclusion chromatography.
  • the liquid chromatography comprises reverse phase chromatography.
  • the liquid chromatography comprises ion exchange chromatography.
  • the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using dialysis.
  • the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using tangential flow filtration.
  • the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide after elongating the oligonucleotide by four or more nucleotides. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide only after elongating the oligonucleotide by four or more nucleotides. In some aspects, the method comprises repeating the method for at least two cycles to further elongate the elongated oligonucleotide without the 3' blocking moiety. In some aspects, the oligonucleotide is at least 4 nucleotides in length.
  • the method comprises making the oligonucleotide prior to elongating the oligonucleotide with the transferase.
  • making the oligonucleotide comprises attaching a nucleotide donor to a nucleotide acceptor having a 3'-OH group using a primase.
  • the nucleotide acceptor is a nucleotide monomer.
  • the nucleotide acceptor is a nucleotide polymer.
  • the nucleotide polymer is a 2-mer, 3-mer, 4-mer, 5-mer, 6-mer, or 7-mer.
  • the nucleotide donor is a nucleotide monomer.
  • the nucleotide donor comprises a 3'-blocking moiety.
  • the 3'-blocking moiety of the nucleotide donor is a phosphate.
  • the method comprises removing the 3'-blocking moiety of the nucleotide donor attached to the nucleotide acceptor to form an elongated nucleotide acceptor.
  • the 3'- blocking moiety is removed from the nucleotide donor using a hydrolase.
  • making the oligonucleotide comprises one or more primase extension cycles, comprising: attaching a nucleotide donor comprising a 3'-blocking moiety to a nucleotide acceptor having a 3'-OH group using a primase to make a 3' -blocked donor-acceptor oligonucleotide; inactivating the primase or separating the primase from the 3' -blocked donor-acceptor oligonucleotide; removing the 3'-blocking moiety from the 3' -blocked donor-acceptor oligonucleotide; wherein each cycle uses a new nucleotide donor.
  • the transferase comprises a single strand RNA ligase.
  • the nucleotide comprising the 3' blocking moiety comprises a conjugate moiety, a reactive group, or a linker.
  • the nucleotide comprising the 3' blocking moiety comprises the conjugate moiety, and the conjugate moiety comprises a carbohydrate, a lipid or a lipophilic group, a sterol, a drug, a hormone, a polymer, a protein, a peptide, a toxin, a vitamin, or a combinations thereof.
  • the oligonucleotide comprises a 5' blocking moiety other than a 5'-phosphate moiety or a 5'- OH.
  • the oligonucleotide is elongated under an inert atmosphere.
  • the inert atmosphere is maintained by an inert gas.
  • the inert gas is argon or nitrogen.
  • elongating the oligonucleotide is performed between 35 °C and 45 °C.
  • removing the 3' blocking moiety is performed between 45 °C and 55 °C.
  • the elongated oligonucleotide without the 3' blocking moiety is buffer exchanged and/or concentrated using a desalting chamber.
  • the desalting chamber is maintained between 20 °C and 30 °C.
  • a method of template-free synthesis of an oligonucleotide comprising: in first reaction chamber, elongating an oligonucleotide in a solution by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide using a transferase immobilized on a solid support suspended in the solution to make an elongated oligonucleotide comprising the 3' blocking moiety; flowing the elongated oligonucleotide comprising the 3' blocking moiety in the solution to a second reaction chamber, wherein the transferase is retained in the first reaction chamber; in the second reaction chamber, removing the 3' blocking moiety from the an elongated oligonucleotide comprising the 3' blocking moiety in the solution using a hydrolase immobilized on a solid support suspended in the solution to make an elongated oligonucleotide without the 3' blocking moiety; and flowing
  • the method further comprises separating unreacted nucleotides or reaction byproducts in the solution from the elongated oligonucleotide without the 3' blocking moiety or the elongated oligonucleotide comprising the 3' blocking moiety.
  • the separating comprises flowing the elongated oligonucleotide comprising the 3' blocking moiety from the first reaction chamber to a purification chamber configured to separate unreacted nucleotides or reaction byproducts from the elongated oligonucleotide comprising the 3' blocking moiety.
  • the separating comprises flowing the elongated oligonucleotide without the 3' blocking moiety from the second reaction chamber to a purification chamber configured to separate unreacted nucleotides or reaction byproducts from the elongated oligonucleotide without the 3' blocking moiety.
  • the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using liquid chromatography.
  • the liquid chromatography comprises size exclusion chromatography.
  • the liquid chromatography comprises reverse phase chromatography.
  • the liquid chromatography comprises ion exchange chromatography.
  • the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using dialysis. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using tangential flow filtration. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide after elongating the oligonucleotide by four or more nucleotides. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide only after elongating the oligonucleotide by four or more nucleotides.
  • the method comprises repeating the method for at least two cycles to further elongate the elongated oligonucleotide without the 3' blocking moiety.
  • the nucleotide comprising the 3' blocking moiety is a preselected type of nucleotide.
  • the hydrolase is a phosphatase. In some aspects, the hydrolase is an alkaline phosphatase. In some aspects, the hydrolase is retained in the second reaction chamber when the elongated oligonucleotide without the 3' blocking moiety flows out of the second reaction chamber.
  • the first reaction chamber and the second reaction chamber are connected to each other through one or more conduits, and the elongated oligonucleotide comprising the 3' blocking moiety and the elongated oligonucleotide without the 3' blocking moiety flows through at least a portion of the one or more conduits.
  • the flowing of the elongated oligonucleotide comprising the 3' blocking moiety to the second reaction chamber and/or the flowing of the elongated oligonucleotide without the 3' blocking moiety to the first reaction chamber is controlled by one or more pumps.
  • the flowing of the elongated oligonucleotide comprising the 3' blocking moiety to the second reaction chamber and/or the flowing of the elongated oligonucleotide without the 3' blocking moiety to the first reaction chamber is controlled by one or more valves.
  • the nucleotide comprising the 3' blocking moiety is a nucleotide triphosphate comprising a 3' blocking moiety or an analog thereof comprising a 5' phosphate analog.
  • the 5' phosphate analog is a 5'-(a- P-thio)phosphate moiety.
  • the hydrolase removes the 3' blocking moiety from unreacted nucleotides in the solution.
  • the hydrolase removes one or more 5' phosphate moieties and/or 5' phosphate analogs from unreacted nucleotides in the solution. In some aspects, the hydrolase removes three 5' phosphate moieties and/or 5' phosphate analogs from unreacted nucleotides in the solution. In some aspects, the hydrolase is immobilized on a solid support. In some aspects, the hydrolase is immobilized using a covalent, electrostatic, or in some aspects, the transferase is a template independent transferase. In some aspects, the transferase is a terminal deoxynucleotidyl transferase (TdT).
  • TdT terminal deoxynucleotidyl transferase
  • the transferase is immobilized on a solid support. In some aspects, the transferase is immobilized using a covalent, electrostatic, or ionic bond. In some aspects, the elongating produces an inorganic pyrophosphate byproduct. In some aspects, the method further comprises degrading the inorganic pyrophosphate using a pyrophosphatase. In some aspects, he pyrophosphatase is an inorganic pyrophosphatase. In some aspects, the pyrophosphatase is immobilized on a solid support. In some aspects, the pyrophosphatase is immobilized using a covalent, electrostatic, or ionic bond.
  • the elongating and the degrading occur within the same reaction chamber.
  • the transferase and the inorganic pyrophosphatase are fused together.
  • the transferase and the pyrophosphatase are immobilized on the same solid support.
  • the transferase and the pyrophosphatase are immobilized on different solid supports.
  • the transferase is immobilized on a solid support and the pyrophosphatase is in the solution.
  • the pyrophosphatase is retained in the reaction chamber comprising the transferase, wherein the reaction chamber comprises a filter that prevents passage of the pyrophosphatase and allows passage of the oligonucleotide .
  • the elongating and the degrading occur within different reaction chambers.
  • the first reaction chamber or the second reaction chamber is a column.
  • the first reaction chamber comprises a fixed bed comprising the transferase immobilized on a solid support.
  • the first reaction chamber comprises a fluidized bed comprising the transferase immobilized on a solid support.
  • the first reaction chamber comprises a filter that prevents passage of the transferase and allows passage of the oligonucleotide.
  • the second reaction chamber comprises a fixed bed comprising the hydrolase immobilized on a solid support.
  • the second reaction chamber comprises a fluidized bed comprising the hydrolase immobilized on a solid support.
  • the second reaction chamber comprises a filter that prevents passage of hydrolase and allows passage of the oligonucleotide.
  • the first reaction chamber or the second reaction chamber is a batch reaction chamber.
  • the batch reaction chamber comprises an impeller.
  • the first reaction chamber comprises a rotating bed reactor comprising the transferase immobilized on a solid support.
  • the second reaction chamber comprises a rotating bed reactor comprising hydrolase immobilized on a solid support.
  • the 3' blocking moiety is a phosphate moiety.
  • the nucleotide comprising the 3' blocking moiety is a ribonucleotide.
  • the nucleotide comprising the 3' blocking moiety is a deoxyribonucleotide.
  • the nucleotide comprising the 3' blocking moiety comprises a 2' modification.
  • the 2' modification is 2'-F or 2'-0Me.
  • the nucleotide comprising the 3' blocking moiety comprises a nucleoside 5'-(a-P-thio)phosphate.
  • the oligonucleotide comprises a 5' modification. In some aspects, the oligonucleotide is at least 4 nucleotides in length. In some aspects, the method comprises making the oligonucleotide prior to elongating the oligonucleotide with the transferase. In some aspects, making the oligonucleotide comprises attaching a nucleotide donor to a nucleotide acceptor having a 3'-OH group using a primase. In some aspects, the nucleotide acceptor is a nucleotide monomer. In some aspects, the nucleotide acceptor is a nucleotide polymer.
  • the nucleotide polymer is a 2-mer, 3-mer, 4-mer, 5-mer, 6-mer, or 7-mer.
  • the nucleotide donor is a nucleotide monomer.
  • the nucleotide donor comprises a 3'-blocking moiety.
  • the 3'-blocking moiety of the nucleotide donor is a phosphate.
  • the method comprises removing the 3'-blocking moiety of the nucleotide donor attached to the nucleotide acceptor to form an elongated nucleotide acceptor.
  • the 3'- blocking moiety is removed from the nucleotide donor using a hydrolase.
  • making the oligonucleotide comprises one or more primase extension cycles, comprising: attaching a nucleotide donor comprising a 3'-blocking moiety to a nucleotide acceptor having a 3'-OH group using a primase to make a 3' -blocked donor-acceptor oligonucleotide; inactivating the primase or separating the primase from the 3' -blocked donor-acceptor oligonucleotide; removing the 3'-blocking moiety from the 3' -blocked donor-acceptor oligonucleotide; wherein each cycle uses a new nucleotide donor.
  • the transferase comprises a single strand RNA ligase.
  • the nucleotide comprising the 3' blocking moiety comprises a conjugate moiety, a reactive group, or a linker.
  • the nucleotide comprising the 3' blocking moiety comprises the conjugate moiety, and the conjugate moiety comprises a carbohydrate, a lipid or a lipophilic group, a sterol, a drug, a hormone, a polymer, a protein, a peptide, a toxin, a vitamin, or a combinations thereof.
  • the oligonucleotide comprises a 5' blocking moiety other than a 5'-phosphate moiety or a 5'- OH.
  • the oligonucleotide is elongated under an inert atmosphere.
  • the inert atmosphere is maintained by an inert gas.
  • the inert gas is argon or nitrogen.
  • elongating the oligonucleotide is performed between 35 °C and 45 °C.
  • removing the 3' blocking moiety is performed between 45 °C and 55 °C.
  • the elongated oligonucleotide without the 3' blocking moiety is buffer exchanged and/or concentrated using a desalting chamber.
  • the desalting chamber is maintained between 20 °C and 30 °C.
  • a method of template-free synthesis of an oligonucleotide comprising: elongating an oligonucleotide in solution by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide using a transferase in the solution to make an elongated oligonucleotide comprising the 3' blocking moiety; separating the elongated oligonucleotide comprising the 3' blocking moiety from the transferase; removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the 3' blocking moiety, the elongated oligonucleotide comprising the 3' blocking moiety being in solution when the 3' blocking moiety is removed.
  • the method comprises separating the elongated oligonucleotide comprising the 3' blocking moiety from the transferase comprises flowing the solution through a column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety.
  • the column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety is a size exclusion column, an affinity column, an ion exchange column, or a reverse phase column.
  • the column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety comprises a filter that prevents passage of the transferase and allows passage of the oligonucleotide comprising the 3' blocking moiety.
  • the method comprises flowing the oligonucleotide and the transferase, in the solution, from a reagent reservoir to the column that retains the transferase, and flowing the oligonucleotide comprising the 3' blocking moiety, in the solution, from the column that retains the transferase to the reagent reservoir.
  • removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety comprises adding a hydrolase to the solution after separating the elongated oligonucleotide comprising the 3' blocking moiety from the transferase.
  • the method further comprises separating the hydrolase from the elongated oligonucleotide without the 3' blocking moiety.
  • separating the hydrolase from the elongated oligonucleotide without the 3' blocking moiety from the hydrolase comprises flowing the solution through a column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety.
  • the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety is a liquid chromatography column. In some aspects, the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety is a size exclusion column, an affinity column, an ion exchange column, or a reverse phase column. In some aspects, the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety comprises a filter that prevents passage of the hydrolase and allows passage of the elongated oligonucleotide without the 3' blocking moiety.
  • the method comprises flowing the elongated oligonucleotide without the 3' blocking moiety and the hydrolase, in the solution, from a reagent reservoir to the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety, and flowing the elongated oligonucleotide without the 3' blocking moiety, in the solution, from the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety to the reagent reservoir.
  • the hydrolase is a phosphatase.
  • the hydrolase is an alkaline phosphatase.
  • the method comprises replacing or cleaning the column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety or the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety.
  • the cleaning comprises flowing a buffer through the column to elute the transferase or the hydrolase.
  • the method further comprises separating unreacted nucleotides or reaction byproducts in the solution from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety.
  • separating unreacted nucleotides or reaction byproducts in the solution from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety comprises flowing the solution through a column that separates the unreacted nucleotides or reaction byproducts from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety.
  • the column that separates the unreacted nucleotides or reaction byproducts from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety is a liquid chromatography column. In some aspects, the column that separates the unreacted nucleotides or reaction byproducts from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety is a size exclusion column.
  • the column that separates the unreacted nucleotides or reaction byproducts from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety is a reverse phase column. In some aspects, the column that separates the unreacted nucleotides or reaction byproducts from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety is an ion exchange column.
  • the unreacted nucleotides or reaction byproducts are separated from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety using dialysis. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety using tangential flow filtration.
  • the unreacted nucleotides or reaction byproducts are separated from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety after elongating the oligonucleotide by four or more nucleotides. In some aspects, the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide only after elongating the oligonucleotide by four or more nucleotides. In some aspects, the nucleotide comprising the 3' blocking moiety is a preselected type of nucleotide.
  • the nucleotide comprising a 3' blocking moiety is a nucleotide triphosphate comprising a 3' blocking moiety or an analog thereof comprising a 5' phosphate analog.
  • the 5' phosphate analog is a 5'-(a-P-thio)phosphate moiety.
  • the hydrolase removes the 3' blocking moiety from unreacted nucleotides in the solution. In some aspects, the hydrolase removes one or more 5' phosphate moieties and/or 5' phosphate analogs from unreacted nucleotides in the solution.
  • the hydrolase removes three 5' phosphate moieties and/or 5' phosphate analogs from unreacted nucleotides in the solution.
  • the transferase is a polymerase from the DNA polymerase X family.
  • the transferase is a template independent transferase.
  • the transferase is a terminal deoxynucleotidyl transferase (TdT).
  • elongating produces an inorganic pyrophosphate byproduct.
  • the method further comprises degrading the inorganic pyrophosphate using a pyrophosphatase.
  • the pyrophosphatase is an inorganic pyrophosphatase. In some aspects, the transferase and the inorganic pyrophosphatase are fused together. In some aspects, the column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety further separates the pyrophosphatase from the elongated oligonucleotide comprising the 3' blocking moiety. In some aspects, the 3' blocking moiety is a phosphate moiety. In some aspects, the nucleotide comprising the 3' blocking moiety is a ribonucleotide.
  • the nucleotide comprising the 3' blocking moiety is a deoxyribonucleotide. In some aspects, the nucleotide comprising the 3' blocking moiety comprises a 2' modification. In some aspects, the 2' modification is 2'-F or 2'-0Me. In some aspects, the nucleotide comprising the 3' blocking moiety comprises a nucleoside 5'-(a-P-thio)phosphate. In some aspects, the oligonucleotide comprises a 5' modification. In some aspects, the oligonucleotide is at least 4 nucleotides in length.
  • the method comprises making the oligonucleotide prior to elongating the oligonucleotide with the transferase.
  • making the oligonucleotide comprises attaching a nucleotide donor to a nucleotide acceptor having a 3'-OH group using a primase.
  • the nucleotide acceptor is a nucleotide monomer.
  • the nucleotide acceptor is a nucleotide polymer.
  • the nucleotide polymer is a 2-mer, 3- mer, 4-mer, 5-mer, 6-mer, or 7-mer.
  • the nucleotide donor is a nucleotide monomer. In some aspects, the nucleotide donor comprises a 3'-blocking moiety. In some aspects, the 3'-blocking moiety of the nucleotide donor is a phosphate. In some aspects, the method comprises removing the 3 '-blocking moiety of the nucleotide donor attached to the nucleotide acceptor to form an elongated nucleotide acceptor. In some aspects, the 3'-blocking moiety is removed from the nucleotide donor using a hydrolase.
  • making the oligonucleotide comprises one or more primase extension cycles, comprising: attaching a nucleotide donor comprising a 3'-blocking moiety to a nucleotide acceptor having a 3'-OH group using a primase to make a 3' -blocked donoracceptor oligonucleotide; inactivating the primase or separating the primase from the 3' -blocked donor-acceptor oligonucleotide; removing the 3'-blocking moiety from the 3' -blocked donor-acceptor oligonucleotide; wherein each cycle uses a new nucleotide donor.
  • the transferase comprises a single strand RNA ligase.
  • the nucleotide comprising the 3' blocking moiety comprises a conjugate moiety, a reactive group, or a linker.
  • the nucleotide comprising the 3' blocking moiety comprises the conjugate moiety, and the conjugate moiety comprises a carbohydrate, a lipid or a lipophilic group, a sterol, a drug, a hormone, a polymer, a protein, a peptide, a toxin, a vitamin, or a combinations thereof.
  • the oligonucleotide comprises a 5' blocking moiety other than a 5'-phosphate moiety or a 5'-OH.
  • the oligonucleotide is elongated under an inert atmosphere.
  • the inert atmosphere is maintained by an inert gas.
  • the inert gas is argon or nitrogen.
  • elongating the oligonucleotide is performed between 35 °C and 45 °C.
  • removing the 3' blocking moiety is performed between 45 °C and 55 °C.
  • the elongated oligonucleotide without the 3' blocking moiety is buffer exchanged and/or concentrated using a desalting chamber.
  • the desalting chamber is maintained between 20 °C and 30 °C.
  • a system for template-free synthesis of an oligonucleotide comprising: a reagent reservoir; a first column configured to substantially separate a transferase from an oligonucleotide comprising a 3' blocking moiety; and a second column configured to substantially separate a hydrolase from an oligonucleotide without the 3' blocking moiety; wherein the system is configured to (i) flow a solution comprising an oligonucleotide comprising the 3' blocking moiety and a transferase from the reagent reservoir to the first column, (ii) substantially separate the oligonucleotide comprising the 3' blocking moiety from the transferase in the first column, (iii) flow the solution comprising the oligonucleotide comprising the 3' blocking moiety from the first column to the reagent reservoir, (iv) flow the solution comprising an oligonucleotide without the 3' blocking moiety and a hydro
  • the system comprises a plurality of conduits that connects the first column, the second column, and the reagent reservoir.
  • the system further comprises a purification chamber configured to separate unreacted nucleotides or reaction byproducts in the solution from the oligonucleotide comprising the 3' blocking moiety or the oligonucleotide without the 3' blocking moiety.
  • the system further comprises one or more conduits that connects the purification chamber and the reagent reservoir.
  • the purification chamber comprises a column.
  • the purification chamber comprises a liquid chromatography column.
  • the purification chamber is a size exclusion column.
  • the purification chamber is an ion exchange column.
  • the purification chamber is reverse phase column. In some aspects, the purification chamber is part of a tangential flow filtration system. In some aspects, the system is further configured to (vii) flow the solution comprising the oligonucleotide comprising the 3' blocking moiety or the oligonucleotide without the 3' blocking moiety from the reagent reservoir to the purification chamber, (viii) separate unreacted nucleotides or reaction byproducts in the solution from the oligonucleotide comprising the 3' blocking moiety or the oligonucleotide without the 3' blocking moiety in the purification chamber, and (ix) flow the solution comprising the oligonucleotide comprising the 3' blocking moiety or the oligonucleotide without the 3' blocking moiety from the purification chamber to the reagent reservoir.
  • the system further comprises one or more diverter valves configured to controllably flow the solution from the reagent reservoir to the first column or the second column.
  • the one or more diverter valves is configured to select a flow path for the solution, wherein the flow path is selected from a plurality of flow paths comprising (i) flow of the solution from the reagent reservoir to the first column and from the first column to the reagent reservoir and (ii) flow of the solution from the reagent reservoir to the second column and from the second column to the reagent reservoir.
  • the system comprises the purification chamber, and the one or more diverter valves is further configured to controllably flow the solution from the reagent reservoir to the purification chamber.
  • the plurality of flow paths further comprises (iii) flow of the solution from the reagent reservoir to the purification chamber and from the purification chamber to the reagent reservoir.
  • the system further comprises one or more wash buffer reservoirs connected to the first column, the second column, or the purification chamber.
  • the one or more diverter valves is configured to controllably flow wash buffer from the wash buffer reservoir to the first column, the second column, or the purification chamber.
  • the one or more diverter valves is configured to select a wash buffer flow path for wash buffer in the one or more wash buffer reservoirs, wherein the flow path is selected from a plurality of flow paths comprising (i) flow of the wash buffer from the one or more wash buffer reservoirs to the first column and from the first column to a system waste outlet and (ii) flow of the wash buffer from the one or more wash buffer reservoirs to the second column and from the second column to the system waste outlet.
  • the system comprises the purification chamber, and wherein the plurality of flow paths further comprises (iii) flow of the wash buffer from the one or more wash buffer reservoirs to the purification chamber and from the purification chamber to the system waste outlet.
  • the system further comprises a temperature regulator that controls a temperature of the solution in the system.
  • the temperature regulator is configured to control the temperature of the solution in the first column or the second column.
  • the system comprises an in-line temperature regulator that controls a temperature of the solution in one or more conduits of the system.
  • the temperature regulator is a jacketed stir tank.
  • the system comprises one or more pumps configured to flow the solution from the reagent reservoir to the first column or from the reagent reservoir to the second column.
  • the system comprises the purification chamber, and the one or more pumps are further configured to flow the solution from the reagent reservoir to the purification chamber.
  • the one or more pumps are configured to control a flow rate of the solution.
  • the first column is further configured to substantially separate a pyrophosphatase from the oligonucleotide comprising a 3' blocking moiety.
  • the system further comprises a third column configured to substantially separate a pyrophosphatase from the oligonucleotide comprising a 3' blocking moiety, wherein the system is further configured to flow a solution comprising the oligonucleotide comprising the 3' blocking moiety and the pyrophosphatase from the reagent reservoir to the third column, (ii) substantially separate the oligonucleotide comprising the 3' blocking moiety from the pyrophosphatase in the third column, and (iii) flow the solution comprising the oligonucleotide comprising the 3' blocking moiety from the third column to the reagent reservoir.
  • the first column comprises a filter that prevents passage of the transferase and allows passage of the oligonucleotide. In some aspects, the first column comprises a resin that binds the transferase. In some aspects, the first column is an ion exchange column, an affinity column, a size exclusion column, or reverse phase column. In some aspects, the second column comprises a filter that prevents passage of the hydrolase and allows passage of the oligonucleotide. In some aspects, the second column comprises a resin that binds the transferase. In some aspects, the second column is an ion exchange column, an affinity column, a size exclusion column, or reverse phase column. In some aspects, the reagent reservoir comprises an impeller.
  • the reagent reservoir comprises a reagent port.
  • the first column or the second column are cleanable or replaceable.
  • the system further comprises a reaction chamber comprising a primase.
  • the primase is immobilized on a solid support.
  • the reaction chamber comprising the primase comprises a filter that prevents passage of the primase and allows passage of a 3' -blocked donor-acceptor oligonucleotide.
  • the system comprises a degassing system or a sparging system.
  • the system comprises a degassing system, and the degassing system comprises a vacuum pump.
  • the system comprises the sparging system.
  • the sparging system is an in-line sparging system or is configured to sparge liquids in a reservoir.
  • the unit for sparging system is configured to sparge using an inert gas.
  • the inert gas is argon or nitrogen.
  • the system comprises a degasser configured to remove oxygen from the system.
  • the purification chamber is configured to concentrate the elongated oligonucleotide comprising the 3' blocking moiety.
  • the purification chamber comprises a membrane with a molecular weight cutoff of about 500 kDa to about 5000 kDa.
  • a composition comprising the oligonucleotide made according to the methods described herein.
  • the oligonucleotide is substantially free of depurination or depyrimidination impurities.
  • the oligonucleotide is substantially free of N-3- cyanoethylthymine (CNET) impurities.
  • the oligonucleotide is substantially free of N(2)-actyl-2,6-diaminopurine and/or an isobutyryl diaminopurine impurities.
  • the oligonucleotide is substantially free of methylcytosine.
  • FIG. l shows a schematic of one cycle of template-free synthesis of an oligonucleotide, according to some aspects.
  • FIG. 2A shows an exemplary rotating bed reactor that may be used in a reaction chamber described herein, according to some embodiments.
  • FIG. 2B shows an exemplary reaction chamber, which is a flow-through column having a fixed bed reactor, in accordance with some embodiments. See Basso et al., Industrial applications of immobilized enzymes - A review, Molecular Catalysis, vol. 479, no. 110607 (2019).
  • FIG. 2C shows another exemplary reaction chamber, which is a flow-through column having a fluidized bed reactor, in accordance with some embodiments.
  • FIG. 2D shows another exemplary reaction chamber, which is a batch reaction chamber having an impeller that can stir contents of the batch reaction chamber, according to some embodiments.
  • This type of reaction chamber may be referred to as a stirred reactor.
  • FIG. 3 shows an exemplary system for template-free synthesis of an oligonucleotide, wherein the system comprises a first container/chamber and a second container/chamber that are fluidly connected, in accordance with some embodiments.
  • the first container/chamber comprises a transferase
  • the second container/chamber comprises a hydrolase.
  • FIG. 4 shows an exemplary system for template-free synthesis of an oligonucleotide, wherein the system comprises a first container/chamber and a second container/chamber that are fluidly connected, and flow is regulated by a control valve, in accordance with some embodiments.
  • the first container/chamber comprises a transferase
  • the second container/chamber comprises a hydrolase.
  • FIG. 5 shows an exemplary system for template-free synthesis of an oligonucleotide, wherein the system comprises a first container/chamber and a second container/chamber that are fluidly connected, and the flow is regulated by a pump, in accordance with some embodiments.
  • the first container/chamber comprises a transferase
  • the second container/chamber comprises a hydrolase.
  • FIG. 6 shows an exemplary system for template-free synthesis of an oligonucleotide, wherein the system comprises a first container/chamber and a second container/chamber that are fluidly connected to each other, in accordance with some embodiments.
  • the flow is regulated by control valves and pumps.
  • the first container/chamber comprises a transferase
  • the second container/chamber comprises a hydrolase.
  • FIG. 7 shows an exemplary system for template-free synthesis of an oligonucleotide, wherein the system comprises a first container/chamber and a second container/chamber that are fluidly connected to each other, in accordance with some embodiments.
  • the flow is regulated by control valves and pumps.
  • the first container/chamber comprises a transferase
  • the second container/chamber comprises a hydrolase.
  • the system comprises a reaction chamber 3, which can comprise an enzyme such as another transferase.
  • FIG. 8 shows an exemplary system for template-free synthesis of an oligonucleotide, wherein the system comprises a first container and a second container that are fluidly connected to each other, in accordance with some embodiments.
  • the flow is regulated by control valves and pumps.
  • the first container comprises a transferase
  • the second container comprises a hydrolase.
  • the system comprises a reagent reservoir that can also act as a product reservoir.
  • FIG. 9 shows an exemplary system for template-free synthesis of an oligonucleotide, wherein the system comprises a first container and a second container that are fluidly connected to each other, in accordance with some embodiments.
  • the flow is regulated by control valves and pumps.
  • the first container comprises a transferase
  • the second container comprises a hydrolase.
  • the system comprises a reagent reservoir that can also act as a product reservoir.
  • One or more reagent reservoirs can be connected upstream of the primary reagent reservoir.
  • FIG. 10 shows an exemplary system for template-free synthesis of an oligonucleotide using chambers, such as in-line reaction chambers with enzymes being retained in the reaction chambers as the synthesized oligonucleotide can flow out of the reaction chambers, in accordance with some embodiments.
  • the oligonucleotide is transferred through the system for extension and cycling using linear flow.
  • FIG. 11 shows an exemplary system for template-free synthesis of an oligonucleotide using reaction chambers such as in-line columns, in accordance with some embodiments.
  • the enzymes are immobilized on a solid support, such as a fluidized bed or a fixed bed.
  • the oligonucleotide is transferred through the system for extension and cycling using flow controlled by a pump.
  • FIG. 12 shows an exemplary system for template-free synthesis of an oligonucleotide using reaction chambers such as stirred reactor, in accordance with some embodiments.
  • the oligonucleotide is transferred through the system for extension and cycling using flow controlled by a pump.
  • FIG. 13 shows an exemplary system for template-free synthesis of an oligonucleotide using a reaction chamber such as a stirred reactor, in accordance with some embodiments.
  • Purification chambers such as in line columns can be used for removing enzyme.
  • the oligonucleotide is transferred through the system for extension and cycling using flow controlled by a pump.
  • FIG. 14 shows an exemplary system for template-free synthesis of an oligonucleotide using reaction chambers such as columns, in accordance with some embodiments.
  • Desalting chambers can be used for separating or removing salts, reagents and other small molecule byproducts from the oligonucleotide.
  • the oligonucleotide is transferred through the system for extension and cycling using flow controlled by a pump.
  • FIG. 15 shows an exemplary system for template-free synthesis of an oligonucleotide using reaction chambers such as columns, in accordance with some embodiments.
  • Desalting chambers can be used for separating or removing salts, reagents and other small molecule byproducts from the oligonucleotide.
  • the oligonucleotide is transferred through the system for extension and cycling using flow controlled by a pump.
  • FIG. 16 shows an impurity profile of an oligonucleotide synthesized by template-free synthesis.
  • the present disclosure relates generally to methods of template-free synthesis of an oligonucleotide, as well as systems related thereto.
  • the systems can be embodied by any of the system designs and/or combination of the system designs as described herein. More specifically, the present disclosure relates to the discovery of systems and methods that enable template-free synthesis, and especially commercial scale template-free synthesis, of oligonucleotides in an efficient and economical manner. Also provided herein are methods that use these systems described herein.
  • Oligonucleotides were previously known to be synthesized in a manner that requires a large, upfront investment in reagents, such organic solvents, to drive the large-scale production at the expense of producing large amounts of chemical waste is also costly to dispose. Additionally, current enzymatic and or chemical methods for oligonucleotide synthesis do not guarantee commercially viable yields. Current methods produce oligonucleotides at lower scales because the process of elongation and extension compromise product quality through degradation, or product yield is reduced because it must be recovered from contaminants and other byproducts that are generated from the synthesis process.
  • oligonucleotide For enzymatic synthesis of oligonucleotides, one such reagent that is limited in quantity and cost prohibitive is enzyme.
  • the inventors have discovered systems and methods that retain the enzyme in their reaction chamber, while the oligonucleotide (e.g., the substrate oligonucleotide) is flowed in solution between chambers and reservoirs. Retaining enzyme in the reaction chamber permits for re-use, decreasing the total quantity of enzyme required for synthesis of oligonucleotide of a predetermined sequence.
  • the oligonucleotides that can be synthesized using these systems and methods are of a predetermined sequence can be made with high target yields and accuracy in solution (e.g., under flow manufacturing conditions), requiring little user intervention between iterative extension cycles and enhancing reagent efficiency, therefore making the oligonucleotides commercially viable as a reagent or active pharmaceutical ingredient.
  • These systems and methods also reduce reagent use and cost and remove the barriers that inhibit large scale oligonucleotide synthesis.
  • the inventors have surprisingly discovered that systems and methods permit large scale production of pre-determined oligonucleotides lengths that are longer compared to what is presently achievable by cunent synthesis techniques in an economic manner.
  • the systems and methods disclosed herein offer a substantial improvement over enzyme mediated oligonucleotide synthesis and chemical methods of oligonucleotide synthesis.
  • oligonucleotide comprising a 3' blocking moiety and a transferase, yielding an elongated oligonucleotide comprising a 3' blocking moiety.
  • the 3' blocking moiety prevents additional addition of nucleotides because it sterically hinders further elongation of the oligonucleotide chain by the transferase.
  • the oligonucleotide comprising the 3' is retained in solution, then is then separated from the enzyme, and deblocked by removing a 3' blocking moiety to produce an elongated oligonucleotide.
  • the deblocking is also conducted with the oligonucleotide in solution.
  • the methods described herein describe a method of template-free synthesis of an oligonucleotide, where the oligonucleotide remains in solution (e.g., the liquid phase).
  • the discovery of the systems and the methods that use the systems as described herein enable efficient and economical template-free synthesis of an oligonucleotide through batch or flow production methods.
  • the elongated oligonucleotide After removal of the 3' blocking moiety from the elongated oligonucleotide, the elongated oligonucleotide is flowed from the second reaction chamber back to the first reaction chamber to re-initiate the cycle of elongation and 3' blocking moiety removal (e.g., an exemplary cycle as in FIG. 1) to further elongate the oligonucleotide by another nucleotide.
  • the cycle of elongation and 3' blocking moiety removal e.g., an exemplary cycle as in FIG.
  • the nucleic acid e.g., oligonucleotide, elongated oligonucleotide comprising a 3' blocking moiety, elongated oligonucleotide
  • the systems and methods described herein comprise at least a third reaction chamber, wherein the elongated oligonucleotide can be further elongated by a second transferase to produce a further elongated oligonucleotide comprising the 3' blocking moiety of the second nucleotide.
  • Disclosed herein are also methods and systems that can be used for methods of template-free synthesis of an oligonucleotide by elongating an oligonucleotide in solution using a transferase in solution, then separating the elongated oligonucleotide from the transferase, and removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the 3' blocking moiety.
  • an oligonucleotide comprising a first reaction chamber that comprises a transferase, and a second reaction chamber that comprises a hydrolase. These reaction chambers are fluidly connected such that an oligonucleotide (such as an oligonucleotide in solution) can be flowed between the chambers.
  • an oligonucleotide such as an oligonucleotide in solution
  • an oligonucleotide can be elongated by a transferase, which attaches a 3' blocking moiety to the oligonucleotide, producing an elongated oligonucleotide comprising a 3' blocking moiety.
  • the elongated oligonucleotide comprising a 3' blocking moiety is then flowed to a second reaction chamber, while the transferase is retained in the first chamber.
  • the elongated oligonucleotide comprising a 3' blocking moiety enters the second chamber, which comprises a hydrolase.
  • the hydrolase removes the 3' blocking moiety from the elongated oligonucleotide comprising a 3' blocking moiety, producing an elongated oligonucleotide.
  • the elongated oligonucleotide can then be flowed from the second chamber back to the first chamber, where it can be further elongated by the transferase that was retained in the first chamber.
  • This cycle can be iterated any number of times with the addition of a predetermined nucleotide comprising a 3' blocking moiety to achieve an oligonucleotide of a predetermined sequence.
  • the reservoir is a reagent reservoir that holds additional reagents necessary for the methods described herein, such as buffers, cofactors, and predetermined nucleotides comprising a 3' blocking moiety.
  • the reservoir is a product reservoir that holds or collects product (e.g., an elongated oligonucleotide) after the 3' moiety is removed from the elongated oligonucleotide comprising a 3' moiety.
  • the product reservoir is fluidly connected to the first reaction chamber or the reagent reservoir.
  • the reagent reservoir also acts as a product reservoir.
  • Disclosed herein are systems that are configured to separate the transferase from the oligonucleotide comprising a 3' blocking moiety; and a second column configured to substantially separate a hydrolase from an oligonucleotide without the 3' blocking moiety.
  • the system is further configured with a reagent reservoir, wherein the solution comprising the oligonucleotides are flowed into said reagent reservoir to substantially separate the nucleic acid (e.g., an elongated oligonucleotide comprising a 3' blocking moiety or an elongated oligonucleotide) from the enzyme (e.g., a hydrolase or a transferase) prior to being flowed to the next reaction chamber.
  • the nucleic acid e.g., an elongated oligonucleotide comprising a 3' blocking moiety or an elongated oligonucleotide
  • enzyme e.g., a hydrolase or a transferase
  • systems that further comprise additional modules that control the flow or regulate system conditions.
  • the systems comprise one or more reaction pumps.
  • the systems comprise one or more valves that selectively control a pathway of the solution in the system.
  • These systems as described herein can be automated or configured to flow the substrate oligonucleotide in solution between chambers while retaining the enzymes (e.g., the hydrolase or transferase) in their respective reaction chambers such that they can be reused in the next iterative cycle of oligonucleotide elongation and removal of the 3' blocking moiety from the elongated oligonucleotide.
  • the systems and methods described herein are also configured retain and enable the re-use of the hydrolase, transferase, and/or pyrophosphatase in the downstream cycles of template- free synthesis of the oligonucleotide.
  • the systems described herein can be automated or partially automated.
  • these systems can be embodied by the systems that are described in further detail herein.
  • these systems described herein can further comprise optional features such as one or more temperature regulators, thermometers, valves, pumps, degassers (such as vacuum pumps), spargers (e.g., an in-line sparger and/or a sparger in a reservoir), and flow regulators.
  • substantially separated refers to dividing, excluding, partitioning, or removing 95% or more of a macromolecular species by percent weight.
  • an enzyme e.g., transferase, hydrolase, pyrophosphatase, etc.
  • oligonucleotide if not more than 5% of the starting quantity is included in the solution after the enzyme has been substantially separated from the oligonucleotide (such as an elongated oligonucleotide comprising the 3' blocking moiety).
  • substantially free refers to a composition that contains less than 5% or less of the identified component (e.g., impurity) by percent weight.
  • a composition that is substantially free of an identified impurity contains 5% or less of that impurity.
  • nucleotide triphosphate refers to a nucleoside with three 5' phosphate groups (or 5' phosphate group analogs) but does not exclude other nucleotides comprising additional phosphate (or phosphate analog) moieties. NTPs may also comprise additional modifications, such as on the 3' position of the ribose sugar (e.g., a 3' phosphate group such that the NTP comprises a 3' phosphate group), on the 2'-position of the ribose sugar (OMe, F, H, MOE), locked and glycine NTPs.
  • additional modifications such as on the 3' position of the ribose sugar (e.g., a 3' phosphate group such that the NTP comprises a 3' phosphate group), on the 2'-position of the ribose sugar (OMe, F, H, MOE), locked and glycine NTPs.
  • NTPs may also be also substituted at any of the 5' phosphate groups to create NTP analogs.
  • a non-bridging oxygen on the a-phosphate may be substituted with a sulfur to make nucleoside 5'-(a-P-thio)triphosphates.
  • NTPs may also be modified to remove a nucleobase to generate an abasic nucleotide.
  • nucleotide analog or “NTP analog” refers to a modified NTP that has been chemically modified and has biosimilar properties and functionality to naturally occurring NTPs.
  • NTP analogs are structurally similar to NTPs.
  • an analog of a NTP can comprise a nucleoside 5 '-(a-P-thio)triphosphate moiety .
  • polynucleotide As used herein, “polynucleotide,” “oligonucleotide,” and “nucleic acid’ ’ are used interchangeably herein and refer to two or more nucleosides or nucleotides that are covalently linked together.
  • the polynucleotide may be wholly comprised of ribonucleotides (i.e., RNA), wholly comprised of 2' deoxyribonucleotides (i.e., DNA), wholly comprised of other synthetic nucleotides or comprised of mixtures of synthetic, ribo- and/or 2' deoxyribonucleotides.
  • the polynucleotides may also include modified nucleotides with substitutions, including 2' substitutions (e.g., 2'-flouro, 2'-O- methyl, 2'-O-methoxyethyl, locked or constrained ethyl modifications, and others known to those skilled in the art). Nucleosides will be linked together via standard phosphodiester linkages or via one or more non-standard linkages, including but not limited to phosphothiolated linkages.
  • substitutions e.g., 2'-flouro, 2'-O- methyl, 2'-O-methoxyethyl, locked or constrained ethyl modifications, and others known to those skilled in the art.
  • Nucleosides will be linked together via standard phosphodiester linkages or via one or more non-standard linkages, including but not limited to phosphothiolated linkages.
  • a polynucleotide will typically be composed of the naturally occurring encoding nucleobases (i.e., adenine, guanine, uracil, thymine and cytosine), it may include one or more modified and/or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc.
  • modified or synthetic nucleobases are nucleobases encoding amino-acid sequences. Nucleobases that are modified or synthetic may comprise any known or hypothetical or future discovered modification or structure that would be recognized by one of skill in the art as a modified or synthetic nucleobase.
  • template-free synthesis refers to synthesis of an oligonucleotide or a polynucleotide without the use of template strand as a guide for synthesis of a complementary oligo or polynucleotide strand.
  • template-free synthesis refers to an iterative process, whereby, successive nucleotides are added to a growing oligo or nucleotide chain or oligonucleotide substrate.
  • Substrate or “reagent” in the context of an enzyme mediated reaction refers to the molecule acted on by the enzyme (e.g., a transferase or a hydrolase).
  • a transferase used in the systems and methods disclosed herein act on a substrate (e.g., an oligonucleotide).
  • a hydrolase used in the systems and methods disclosed herein act on a substrate (e.g., an elongated oligonucleotide comprising a 3' blocking moiety or an unreacted nucleotide such as a nucleotide comprising a 3' blocking moiety).
  • a pyrophosphatase used in the systems and methods disclosed herein act on a substrate (e.g., inorganic pyrophosphate).
  • ‘Product” in the context of an enzyme mediated reaction refers to the molecule resulting from the action of the enzyme.
  • the product is the target molecule.
  • an exemplary product for a transferase used in the systems and methods described herein is an elongated oligonucleotide comprising a 3' blocking moiety.
  • “Byproduct” in the context of an enzyme mediated reaction is a secondary product that is generated in addition to the product resulting from the action of the enzyme.
  • byproducts can include, but are not limited to, nucleosides, NDPs, residual NDPs comprising a 3' blocking moiety, NMP, residual NMPs comprising a 3' blocking moiety, phosphate, and residual pyrophosphate.
  • Phosphate refers to a functional group comprised of an orthophosphate ion (phosphorous atom covalently linked to four oxygen atoms).
  • the orthophosphate ion is commonly found with one or more hydrogen atoms or organic groups.
  • a phosphate group or chain may be modified, as further described herein.
  • Phosphorylated refers to the addition or presence of one of more phosphoryl groups (phosphorous atom covalently linked to the three oxygen atoms).
  • reaction chamber for example without limitation, flowing of reaction solution from a reaction chamber to another reaction chamber, or a reaction chamber to a reservoir
  • conduits such as tubes or channels.
  • a reaction chamber in fluid communication with a reservoir allows for flowing of reaction solution between the reaction chamber and the reservoir such as through a conduit.
  • the flow of solution between the elements can be regulated, such as by valves or diverters, as described herein.
  • nucleotide comprises a sugar moiety, which can be further modified at different positions.
  • modified nucleotides contemplated include nucleotides with blocking groups on the 3' positions of the sugar (e.g., nucleotides comprising a 3' blocking moiety), as well as nucleotides with modified bases or thiol derivates for the formation of more stable oligonucleotide phosphorothioate backbone bonds.
  • the blocking group also known to those skilled in the art as an inhibitor or reversible terminating group, may include a variety of groups that prevent the transferase from adding additional nucleotides to the oligonucleotides. This may include charged molecules, large molecules and moieties, or other blocking groups known to those skilled in the art. Appropriate removable blocking groups may include carbonitriles, phosphates, carbonates, carbamates, esters, ethers, borates, nitrates, sugars, phosphoramidates, phenylsulfenates, and sulfates. Other 3' blocking groups are also known in the art, including 3'-0-amines and methylamines. Nucleotides comprising a 3' blocking moiety
  • the systems and methods provided herein can be used to elongate an oligonucleotide in solution (e.g., in a liquid) by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide, for example, by using a transferase to make an elongated oligonucleotide comprising the 3' blocking moiety.
  • the 3' blocking moiety can then be removed from the elongated oligonucleotide comprising the 3' blocking moiety, producing an elongated oligonucleotide.
  • the nucleotide comprising a 3' blocking moiety may comprise a nucleotide triphosphates (NTP).
  • NTP nucleotide triphosphates
  • the NTP further comprises a 3' blocking moiety (e.g., a blocking moiety on the 3' position of the sugar of the nucleotide).
  • a NTP does not exclude the inclusion of other phosphates (e.g., phosphates introduced in the 3' blocking moiety).
  • the nucleotide comprising a 3' blocking moiety comprises a nucleoside tetraphosphate.
  • NTPs with a phosphate group at the 3' position of the sugar are useful for the systems and methods of template-free synthesis as provided herein.
  • An exemplary process of converting a NTP into a NTP with a phosphate group at the 3' position of the sugar is depicted in Scheme 1.
  • the NTP is converted by a 3'0-kinase to a nucleoside tetraphosphate (NQP or pppNp) with the fourth phosphate group at the 3' position of the sugar, thereby generating a nucleotide comprising a 3' blocking moiety.
  • the group R at the 2' position of the sugar (“2'-R group”) may be an atom or group selected from H, OH, OCHj, OCH2CH2OCH3, F, and CO2R’ (where R’ is any alkyl or aryl), or another atom or chemical group. Additionally, the sugar may have other modifications at other positions.
  • the nucleobase may be a uridine, thymine, cytosine, adenine, guanine or another nucleobase known to those skilled in the art.
  • the nucleotide comprising a 3' blocking moiety may also have modifications of the nucleobase or of the 5' phosphate chain.
  • the nucleotide comprising a 3' blocking moiety may comprise one or more modifications.
  • the nucleotide comprising a 3' blocking moiety may comprise ribonucleosides, deoxyribonucleosides, dideoxynucleosides, or modified nucleosides.
  • nucleotide comprising a 3' blocking moiety may comprise one or more modifications to the sugar. In some aspects, nucleotide comprising a 3' blocking moiety may comprise one or more modifications to the nucleobase.
  • the nucleotide comprising a 3' blocking moiety has at the 2'-position of the sugar moiety a H or OH.
  • the nucleobase of the nucleotide comprising a 3' blocking moiety is adenine, cytosine, guanine, thymine, uracil, xanthine, hypoxanthine, 2,6-diaminopurine, purine,
  • the nucleotide comprising a 3' blocking moiety has a nitrogenous base that is adenosine (A), guanosine (G), uridine (U), cytidine (C), or thymidine (T), and wherein the nucleoside has at the 2'-position of the sugar moiety an OH.
  • the nucleotide comprising a 3' blocking moiety is a ribonucleotide.
  • the nucleotide comprising a 3' blocking moiety has a nitrogenous base that is adenosine (A), guanosine (G), uridine (U), cytidine (C), or thymidine (T), and wherein the nucleoside has at the 2'-position of the sugar moiety a H.
  • the nucleotide comprising a 3' blocking moiety is a deoxyribonucleotide.
  • the nucleotide comprising a 3' blocking moiety comprises a modified nucleoside.
  • the nucleoside has a modified sugar moiety or modified nucleobase, or combination thereof.
  • the nucleotide comprising a 3' blocking moiety comprises a modified 2'- position of the sugar moiety (e.g., a 2' modification).
  • the 2' modification is halo, 2'- O-R’, or 2'-O-COR’, where R’ is an alkyl, alkyloxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, arylalkyl, or heteroarylalkyl.
  • R’ is a Cl-C4alkyl.
  • the modified 2'-position is a 2'-O-R’, wherein in R' is alkyloxy alkyl, alkylamine, cyanoalkyl, or -C(O)-alkyl.
  • the 2'-position of the sugar moiety of the nucleoside substrate is -O-R’, wherein R’ is -CH3 or -CH2CH3 or -CH2CH2OCH3.
  • the modified 2'-position is 2'-O-(2-methoxyethyl), 2'-O-ally 1, 2'-O-propargyl, 2'-O-ethylamine, 2'-O- cyanoethyl, or 2-O-acetate ester.
  • the 2'-position of the sugar moiety is halo. In some aspects, the 2'-position of the sugar moiety is F (i.e., 2'-F) or Br (i.e., 2'-Br). In some aspects, the 2' modification is 2'-F. In some aspects, the 2' modification is 2'-OMe.
  • the nucleotide comprising a 3' blocking moiety may comprise an a- thiophosphate or dithiophosphate or other modification to the 5' phosphate chain.
  • the nucleotide comprising a 3' blocking moiety comprises a nucleoside 5'-(a-P-thio)phosphate.
  • a nucleotide that comprises a nucleoside 5'-(a-P-thio)phosphate is also known as an a-phosphate modified nucleotides (e.g., dNTPaS and NTPaS) and are presently used to prepare phosphorothiolate DNAs and RNAs, respectively.
  • a phosphorothiolate bond introduces a sulfur atom in place of a non- briding oxygen in the phosphate backbone.
  • Oligonucleotides comprising phosphorothiolate bonds are more resistant to degradation by nucleases.
  • the present systems and methods as disclosed herein can utilize nucleotides comprising a 3' blocking moiety, and further comprising a 5 '-(a-P-thio)phosphate to elongate an oligonucleotide.
  • nucleotide comprising a 3' blocking moiety comprises a modified nucleobase.
  • the modified nucleobase of the nucleoside substrate is 5-bromo-uracil,
  • the nucleotide comprising a 3' blocking moiety comprises a nucleobase that is, among others, 5-methylcytosine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5- alkylcytidines, 5-alkyluridines, 5-halouridines,
  • 6-azapyrimidines 6- alkylpyrimidines, 5-propynyl-uracil, 2-thio-5-propynyl-uracil, quesosine, 2-thiouridine, 4-thiouridine, 4-acetylcytidine, 5- (carboxyhydroxymethyl)uridine, 5-carboxymethylaminomethyl-2-thiouridine,
  • the nucleotide comprising a 3' blocking moiety comprises a noncanonical nucleobase, a removable tag, a cleavable linker, or a radio, a photo, and/or a chemical sensor.
  • the nucleotide comprising a 3' blocking moiety further comprises a conjugate moiety.
  • the conjugate moiety is on the nucleobase of the nucleotide.
  • the nucleotide comprising a 3' blocking moiety further comprises a 2' conjugate moiety.
  • the 2' conjugate moiety is a ligand.
  • the conjugate moiety i.e., non-nucleotide moiety
  • the conjugate moiety is used to affect the pharmacokinetics of the oligonucleotide and/or oligonucleotide cell targeting.
  • the conjugate moiety can be attached to the 5’ -terminal nucleotide, the 3’ -terminal nucleotide, or in a polynucleotide or oligonucleotide an internal nucleotide.
  • the conjugate moiety is attached the 2’-position of the sugar moiety of a nucleoside, for example, to the 2’ -OH.
  • the conjugate moiety is attached to the 3’ -position of the sugar moiety of the nucleoside, for example 3’ -OH.
  • the conjugate moiety is attached to the nucleobase, as discussed above (see, e.g., Biscans et al., Nucleic Acids Res. 2019 Feb 20; 47(3): 1082-1096). In some embodiments, the conjugate moiety is attached directly or attached using a linker.
  • the conjugate moiety comprises a C6-C22 alkyl, C6-22 alkenyl, or C6-C22 alkynyl.
  • the conjugate moiety comprises a Ce-alkyl, Ck-alkyl. Ck-alkyl, Cg- alkyl, Cio-alkyl, Cn-alkyl, Ci2-alkyl, Ci3-alkyl, Cu-alkyl, Ci5-alkyl, Ci6-alkyl, Cn-alkyl, Cis-alkyl, Cig-alkyl, C2o-alkyl, C2i-alkyl, or C22-alkyl.
  • the conjugate moiety comprises a Cg alkenyl, C7 alkenyl, Ck alkenyl Cg alkenyl, C10 alkenyl, Cn-alkenyl, Ci2-alkenyl, Cn-alkenyl, C14- alkenyl, Cis-alkenyl, Ci6-alkenyl, Cn-alkenyl, Cn-alkenyl, Cig-alkenyl, C2o-alkenyl, C21 -alkenyl, or C22-alkenyl.
  • the conjugate moiety comprises a Cr, alkynyl, C7 alkynyl, Cs alkynyl, Cg alkynyl, C10 alkynyl, Cn-alkynyl, Ci2-alkynyl, Cn-alkynyl, Cn-alkynyl, Cn-alkynyl, C16- alkynyl, Cn-alkynyl, Cn-alkynyl, Cig-alkynyl, C2o-alkynyl, C2i-alkynyl, or C22-alkynyl.
  • the conjugate moiety comprises a heteroalkyl, heteroalkenyl, or heteroalkynyL
  • the heteroalkyl, heteroalkenyl or heteroalkynyl has one or more carbon atoms replaced with a heteroatom, such as O, S, or N.
  • the conjugate moiety comprises a cycloalkyl or heterocycloalkyl group.
  • the cycloalkyl includes, by way of example and not limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3 -cyclohexenyl, and cycloheptyl.
  • the heterocycloalkyl includes, among others, l-(l,2,5,6-tetrahydropyridine, 1- piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, and 2-piperazinyl.
  • the conjugate moiety comprises an aryl or heteroaryl moiety.
  • the aryl group includes, by way of example and not limitation, phenyl, naphthyl, indenyl, biphenyl, phenanthrenyl, naphthacenyl, anthracenyl, fluorenyl, indenyl, and azulenyl.
  • a heteroaryl group includes, among others, pyridyl, furanyl, thienyl, pynolyl, oxazolyl, oxadiazolyl, imidazolyl ihiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isoihiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, isoquinolinyl, and indazolyl.
  • the conjugate moiety comprises a cycloalkylalkyl-, heterocycloalkylalkyl-, arylalkyl-, heteroarylalkyl-, cycloalkylheteroalkyl- heterocycloalkylheteroalkyl-, arylheteroalkyl-, heteroarylheteroalkyl-, cycloalkylalkenyl-, heterocycloalkylalkenyl-, arylalkenyl-, heteroarylalkenyl-, cycloalkylheteroalkenylheterocycloalkylheteroalkenyl-, arylheteroalkenyl-, or heteroarylheteroalkenyl- groups.
  • the conjugate moiety comprises a lipid or lipophilic moiety, for example a fatty acid.
  • the fatty acid comprises a saturated fatty acid, unsaturated fatty acid, or a polyunsaturated fatty acid.
  • the fatty acid comprises caprylic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, elaidic acid, cis-vaccenic acid, trans-vaccenic acid, linoleic acid, alpha-linoleic acid, gammalinoleic acid, arachidonic acid, eicosapentaenoic acid, decanoic acid, docosahexaenoic acid (DHA), and docosanoic acid (DCA) conjugate moieties (see, e.g., Kubo et al., ACS Chem. Biol., 2021, 16, 150-164; see also, W02024/040041; incorporated herein by reference).
  • DCA docosanoic acid
  • the conjugate moiety comprises a sterol.
  • the sterol comprises cholesterol, alpha-cholesterol, cholesterol ester (e.g., cholesteryl palmitate, etc.), cholesterol sulfate, phytosterol, cholic acid, or lithocholic acid.
  • the conjugate moiety comprises a vitamin or vitamin derivative, including, by way of example and not limitation, folate, tocopherol, retinoic acid, vitamin D, and the like (see, e.g., US Patent No. 9789197).
  • the conjugate moiety comprises a phospholipid.
  • the phospholipid comprises phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, or a sphingolipid.
  • the conjugate moiety comprises a carbohydrate, particularly a carbohydrate moiety acting as a ligand for a cellular receptor for cellular targeting of the oligonucleotide.
  • the carbohydrate moiety comprises galactose or galactose derivatives.
  • the carbohydrate moiety is attached to the nucleoside via a linker.
  • carbohydrates moiety include the following:
  • the conjugate moiety is an N-acetylgalactosamine (GalNAc) conjugate moiety.
  • the oligonucleotide acceptor and/or nucleotide donor may be conjugated to at least one conjugate moiety comprising at least one N-acetylgalactosamine (GalNAc) moiety.
  • the conjugate moiety is monovalent, divalent, trivalent or tetravalent, GalNAc.
  • the GalNAc moiety has the following structure,
  • L is a linker
  • W is a heteroatom, such as O or S.
  • the W is the 2’ -OH of the sugar moiety of a nucleoside.
  • An exemplary monovalent GalNAc moiety is
  • the monovalent GalNAc is attached via the linker to the 2’ -position of a nucleoside, such as adenine or guanine.
  • a nucleoside such as adenine or guanine.
  • conjugate moieties can be present in contiguous nucleotides in a polynucleotide or oligonucleotide (see, e.g., W02024/040041).
  • the conjugate moiety is a trivalent GalNAc.
  • Tri- valent N- acety [galactosamine conjugate moieties are described in, for example, International patent publication WO 2014/076196, WO 2014/207232 and WO 2014/179620.
  • “Trivalent GalNAc” refers to a residue comprising three N-acetylgalactosamine moieties, typically attached via a linker.
  • An exemplary trivalent GalNAc conjugate moieties are depicted below:
  • the conjugate moiety comprises a reporter molecule.
  • reporter molecules include, among others, fluorescent moieties, such as fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxy- fluorescein, 6-carboxyfluorescein or FAM), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy -X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine Green, rhodamine
  • fluorescent moieties such as
  • the reporter moiety is a chemiluminescent moiety, for example acridinium esters, ruthenium derivatives (e.g., tris(2,2'-bipyridyl) ruthenium), and dioxetanes.
  • acridinium esters for example acridinium esters, ruthenium derivatives (e.g., tris(2,2'-bipyridyl) ruthenium), and dioxetanes.
  • the conjugate moiety comprises an affinity or capture tag.
  • affinity or capture tag includes, among others, biotin, desthiobiotin, digoxigenin, 3-amino-3- deoxydigoxigenin, and a hapten (e.g., dinitrophenol, Alexa Fluor 40, Alexa Fluor 488, dansyl, Lucifer yellow, Oregon Green 488, fluorescein).
  • the conjugate moiety comprises a peptide.
  • the peptide comprises a cellular targeting peptide and/or cell penetration peptide (CPP) for enhancing cellular delivery of a conjugate modified oligonucleotide.
  • CPP cell penetration peptide
  • the cell penetrating peptide is attached via a linker, including a cleavable linker.
  • Cell penetrating peptides include among others, TAT, penetratin, MAP, transportan/TPIO, VP22, polyarginine, MPG, Pep-1, pVEC, YTA2, YTA4, M918, and CADY.
  • the conjugate moiety comprises an RGD (Arg- Gly-Asp) peptide. Sequences of some penetrating peptides are described in Copolovici et al., 2014, 8(3): 1972-1994 and some are provided below: [0108] Other cell penetrating peptides, including those conjugated to nucleic acids, are disclosed in, among others, patent publications WO24063570, WO24044663, US2024083949, WO24026141, W023230600, WO23219933, WO23177261, WO23178327, WO23093960, WO23086342, WO23081893, WO23069332, W023070108, WO23034515, US2023248630, US2023053924, W023003380, WO23277628, WO23277575, US2022378946, WO22171972, W022162200, WO2020144233, WO22180242, WO22132
  • the nucleotide comprising a 3' blocking moiety further comprises a targeting moiety.
  • the nucleotide comprising a 3' blocking moiety further comprises a reactive moiety.
  • the reactive moiety is on the nucleobase of the nucleotide.
  • the nucleotide comprising a 3' blocking moiety further comprises a 2' reactive moiety.
  • the reactive group is attached to the nucleoside via a linker.
  • the reactive group is a cyano, azido, alkynyl, amino, carboxyl, sulfhydryl, dibenzocyclooctynyl, vinyl, trans-cyclooctene, or tetrazine.
  • the reactive group is those used for click chemistry, including copper free click chemistry. Exemplary reactive groups are provided below:
  • the conjugate moiety or reactive moiety is attached to the nucleoside or the terminal group through a linker.
  • linkers are known in the art for conjugating chemical groups to nucleosides and phosphate groups.
  • Linkers can be, among others, substituted or unsubstituted alkylene, heteroalkylene, alkenylene, heteroalkenylene, arylene, heteroarylene, arylalkylene, arylalkenylene, heteroarylalkylene, heteroarylalkenylene, arylheteroalkylene, arylheteroalkenylene, heteroarylheteroalkylene, and heteroarylalkenylene.
  • the linker comprises substituted or unsubstituted C2-C22 alkylene, heteroalkylene, or polyethylene glycol.
  • the linkers have functional groups for conjugation.
  • the linker L has the structure below:
  • the linker comprises a substituted or unsubstituted polyethylene glycol linker.
  • the polyethylene glycol linker has the formula:
  • n is 2-24. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
  • the polyethylene glycol linker has the structure below:
  • exemplary polyethylene glycol linkers have the structure below:
  • the linker is a cleavable linker in which the linker can be cleaved, for example to detach a conjugate moiety.
  • a cleavable linker includes, by way of example and not limitation, a disulfide linkage, enzymatically cleavable linkers (e.g., peptide linkers), and photocleavable linkers (see, e.g., Hermanson, G., Bioconjugate Techniques, 3rd Ed., 2013, Academic Press; see also Bioconjugation Protocols: Strategies and Methods, In Methods in Molecular Biology, 2 nd Ed., S.S. Mark edminister 2011, Humana Press).
  • bifunctional linkers can be used to attach a conjugate moiety to the linker and attach the linker-conjugate to the nucleoside or vice versa (see, e.g., Hermanson, G., supra; see also Bioconjugation Protocols: Strategies and Methods, In Methods in Molecular Biology, supra).
  • an activating group can be attached to an atom to activate the atom to form a covalent bond with another reactive group. Examples of synthetic activating groups that can be attached to an oxygen atom include, but are not limited to, acetate, succinate, triflate, and mesylate.
  • the activating group can be a group that is derivable from a known coupling reagent.
  • coupling reagents include, but are not limited to, N,N'-dicyclohexylcarbodimide (DCC), hydroxybenzotriazole (HOBt), N-(3-dimethylaminopropyl)-N'-ethylcarbonate (EDC), (denzotriazol- 1 - yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol- 1-yl- oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) or O-benzotriazol-l-yl-N,N,N',N'- tetramethyluronium hexafluorophosphate (HBTU).
  • DCC N,N'-dicyclohexylcarbodimide
  • HOBt hydroxybenzotriazo
  • the modified nucleotides that may be used by the methods and systems as disclosed herein may comprise targeting moieties, such as targeting moieties that have therapeutic relevance.
  • the modified nucleotides may comprise reactive and/or sterically bulky side chains.
  • the nucleotides that can be used for the systems and methods as described herein may be modified as shown herein.
  • the systems and methods provided herein may start from an initiating nucleotide (e.g., an initiating nucleotide acceptor) to generate oligonucleotides that can be elongated in solution (e.g., in a liquid) by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide, for example, by using a transferase to make an elongated oligonucleotide comprising the 3' blocking moiety.
  • an initiating nucleotide e.g., an initiating nucleotide acceptor
  • an initiating nucleotide e.g., an initiating nucleotide acceptor
  • terminal deoxynucleotidyl transferases TdTs
  • poly(N) polymerase TdTs
  • DNA polymerases such as polymerase > require initiating oligonucleotides, preferably 4- 6 nucleotides in length.
  • Primase is an example of a transferase which is a specialized polymerase that can use nucleotides to generate oligonucleotides that can be further elongated using the systems and methods provided herein.
  • the initiating nucleotide is a NTP, NDP, NMP, or a nucleoside. In some aspects, the initiating nucleotide comprises a 5' blocking moiety. In some aspects, the initiating nucleotide is a NTP, NDP, NMP, or a nucleoside that comprises a 5' blocking moiety. As a nonlimiting example, a 5' blocking moiety may be used to inhibit primase from using the nucleotide acceptor as the nucleotide donor. In some aspects, the 5' blocking moiety is a 5' OH. In some aspects, the initiating nucleotide (e.g., an initiating nucleotide acceptor) is an NTP, which can further serve as a nucleotide donor. Reaction Chambers
  • reaction chambers are vessels in which an enzymatic reaction occurs. More specifically, reaction chambers are vessels or containers. Reaction chambers are made of an inert material and are suited for holding solutions, liquids, and/or reagents during enzymatic reactions or processes.
  • the reaction chamber comprise buffers and/or solutions that may be degassed (e.g., using an vacuum pump) and/or sparged (e.g., an in-line sparger or a reservoir that is operably linked to a sparger or tank configured to flow an inert gas through the buffer and/or solution before or during operation of the system.
  • Exemplary inert gases include argon and nitrogen.
  • the enzymatic reaction or process uses multiple reaction chambers before the enzymatic reaction or process is complete. In some aspects, the enzymatic reaction or process is completed in one reaction chamber. Examples of a reaction or process include, but are not limited to, elongating an oligonucleotide in solution using a transferase and removing the removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide using a hydrolase.
  • reaction chambers contemplated herein enable, promote, maintain, and/or retain favorable conditions for controlled enzymatic reactions or processes (e.g., elongating an oligonucleotide in solution using a transferase, removing the removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the 3' blocking moiety using a hydrolase).
  • controlled enzymatic reactions or processes e.g., elongating an oligonucleotide in solution using a transferase, removing the removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the 3' blocking moiety using a hydrolase.
  • Reaction chambers contemplated for use include, but not limited to, reaction chambers fabricated from inert or non-reactive plastics, glass, or inert or non-reactive metals.
  • the reaction chambers are coated.
  • the coating comprises one or more polymers.
  • the coating is hydrophilic.
  • the reaction chambers are sterile. In some aspects, the reaction chambers are reusable. In some aspects, the reaction chambers are cleanable and/or replaceable. In some aspects, the reaction chambers are single-use.
  • the reaction chambers can be of varying sizes and volumes. In some aspects, reaction chambers are suitable for commercial scale production of oligonucleotides. In some aspects, the reaction chambers are 1 mL to 50 mL in volume. In some aspects, the reaction chambers are 50 mL to 250 mL in volume. In some aspects, the reaction chambers are 30 mL to 150 mL in volume. In some aspects, the reaction chambers are 500 mL to 10 L in volume. In some aspects, the reaction chambers are 1 L to 10 L in volume.
  • the reaction chambers are about 5 mL, about 10 mL, about 15 mL, about 50 mL, about 100 mL, about 300 mL, about 500 mL, or about 1 L in volume. In some aspects, the reaction chambers are about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L in volume. In some aspects, the reaction chambers are about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L in volume.
  • the reaction chamber is insulated.
  • the reaction chamber is temperature regulated.
  • the temperature regulation can be achieved by, for example, water recirculation, a thermometer, a heating element, a jacketed reservoir (e.g., a jacketed reagent reservoir such as ajacketed stir tank), a jacketed chamber (e.g., any of the chambers as described herein), or a cooling element.
  • the reaction chamber is maintained at a temperature.
  • the temperature of the reaction chamber is 25 °C, about 30 °C, about 40 °C, about 50 °C, about 60 °C, about 65 °C, about 70 °C, about 75 °C, about 80 °C, about 85 °C, about 90 °C, or about 95 °C. In some aspects, the temperature of the reaction chamber is about 40 °C, about 41 °C, about 42 °C, about 43 °C, about 44 °C, about 45 °C, about 46 °C, about 47 °C, about 48 °C, about 49 °C, or about 50 °C.
  • the temperature of the reaction chamber is 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, or 95 °C.
  • the temperature of the reaction chamber is 40 °C.
  • the first reaction chamber comprising the transferase is about 30°C to about 50 °C (such as about 40 °C).
  • the temperature of the reaction chamber is 50 °C.
  • the temperature of the second reaction chamber (e.g., containing the hydrolase) is about 40 °C to about 60 °C (e.g., about 50 °C).
  • the reaction chambers further comprise an apparatus that can be used to stir or mix the solution.
  • the apparatus is an impeller.
  • the apparatus comprises a magnetic stir bar and a stir plate.
  • apparatus is a rod, such as a glass rod.
  • reaction chambers are vessels that can be adapted for flow manufacturing.
  • the reaction chambers are batch reaction chambers (any suitable for batch reaction) .
  • Non-limiting examples of the reaction chambers are beakers, drum, conical tubes, microfuge tubes, Erlenmeyer flasks, welled plates, and culture flasks.
  • the reaction chambers can be round bottom, flat bottom, baffled bottom, or conical bottom vessels.
  • Reaction chambers may be cylindrical, spherical, or flask shaped.
  • the vessels typically associated with batch processing can be applied to flow manufacturing methods, for example, by using adapters such as rubber stoppers with tubing.
  • a non-limiting example for using a batch vessel for the systems and processes described herein is adapting an Erlenmeyer flask using a rubber stopper and tubing to form an inlet and an outlet.
  • the batch reaction chamber comprises an apparatus for stirring or mixing the solution. Stirring or mixing ensures even distribution of reagents and maintenance of homogeneous conditions throughout the solution. Stirring or mixing is useful for maintaining proper reaction conditions such as by enhancing heat transfer (e.g., preventing heat build up in certain areas of the reaction chamber), promoting buffer and phase homogeneity and uniform distribution of reactants. These are all factors that influence enzyme reaction kinetics and product yield.
  • the apparatus for stirring or mixing is an impeller.
  • the batch reaction chamber comprises one or more impellers. Impellers are rotating components that can be used to mix the solution in a batch reaction chamber. An impeller is a mechanical agitator and can have different geometries.
  • the impeller is a jet impeller, a blade impeller, a propeller, a paddle or a turbine impeller.
  • the apparatus for stirring or mixing the solution is a rotator or a shaker.
  • a rotator or shaker physically rotates or rocks the batch vessel to create movement in the solution.
  • the apparatus for stirring or mixing the solution is a stir bar and a stir plate.
  • a magnetic stir bar is placed inside of batch reaction chamber and placed on top of a magnetic stir plate, which uses a rotating magnetic field to cause the stir bar to spin or rotate at a set speed.
  • Rotating bed reactors are hollow cylinders with a basket inside that is packed with a solid phase or material, such as a packed bed.
  • the basket is immersed in a solution (e.g., a liquid phase or a fluid phase) and spun at a rate to create a flow throughout the basket.
  • Rotating bed reactors are effective for maintaining homogeneous reaction conditions and confer properties such as efficient heat transfer.
  • the packed bed comprises immobilized enzyme on a solid support, such as any of the enzymes described herein.
  • the immobilized enzyme is a transferase or a hydrolase.
  • the immobilized enzyme is a pyrophosphatase. In some aspects, the immobilized enzyme is a transferase, and the transferase is fused to a pyrophosphatase. In some aspects, the packed bed comprises two or more immobilized enzymes. In some aspects, the two or more immobilized enzymes are at least one transferase and at least one pyrophosphatase.
  • reaction chambers contemplated for use in the systems and methods disclosed herein include columns (such as those shown in FIG. 2B-2D). Columns are typically manufactured from a rigid, inert material. Examples include glass columns (e.g., chromatography columns) or columns made of inert plastic. Columns useful for the systems and methods described herein include, but are not limited to, columns from 5-500 cm in length and 0.5 to 10 cm in width. Columns may be capped and operably linked such that they are fluidly connected. The columns may be packed with resin or beads to create a bed. In some aspects, the resin or beads are coupled to enzyme. In some aspects, the enzyme is a transferase. In some aspects, the enzyme is hydrolase.
  • the enzyme is a pyrophosphatase.
  • the enzyme is a transferase fusion, such as a transferase fused to a pyrophosphatase.
  • the enzyme is a transferase-pyrophosphatase fusion. Solutions can migrate through the bed packed in the column via gravity flow or be flowed through using an apparatus that controls flow rate, such as a peristaltic pump.
  • the column comprises a bed that can be a fixed bed (e.g., a packed bed) as shown in FIG. 2B.
  • the bed can be fluidized as shown in FIG. 2C.
  • a fluidized bed is when particulates or solids are suspended in solution such that the bed has fluid-like properties.
  • the column does not comprise a bed or solid support.
  • the column is used as a vessel to hold the solution.
  • the column comprises free enzyme, such as free transferase (e.g., transferase in solution) or free hydrolase (e.g., hydrolase in solution).
  • the enzyme is retained in the column by a porous medium such as a membrane or a filter.
  • the pores of the membrane or filter comprise pores of a determined diameter.
  • the pores of the membrane or filter have a specific molecular weight cut off (MWCO) value. MWCO permits molecules below a specific size to pass through but retains molecules larger than the cut off size.
  • MWCO molecular weight cut off
  • the column comprises a filter that prevents passage of the transferase and allows passage of the oligonucleotide. In some aspects, the column comprises a membrane that prevents passage of the transferase and allows passage of the oligonucleotide. In some aspects, the column comprises a filter that prevents passage of the pyrophosphatase and allows passage of the oligonucleotide.
  • the systems and methods disclosed herein comprise a reaction chamber comprising a transferase, and another reaction chamber comprising a hydrolase.
  • the reaction chambers are batch reaction chambers that optionally include an impeller.
  • one or more of the reaction chambers comprise a rotating bed reactor.
  • the rotating bed reactor is packed with a solid support.
  • the transferase is immobilized on a solid support.
  • the hydrolase is immobilized on a solid support.
  • the reaction chambers are columns, such as fixed bed reactors (see FIG. 2B) or fluidized bed reactors (see FIG. 2C).
  • the reaction chambers are stirred reactors and comprise at least one impeller (see, for example, FIG. 2D).
  • the reaction chambers can be cleanable or replaceable.
  • the reaction chambers comprises an inlet and an outlet.
  • the reaction chambers are fluidly connected by one or more conduits.
  • a conduit such as a conduit that fluidly connects two reaction chambers, may be used to operably link two reaction chambers.
  • the one or more conduits operably links two or more reaction chambers.
  • Non-limiting examples of conduits include chromatography tubing of any material, including glass, plastic, rubber, and silicone.
  • the conduit may be rigid, semi-rigid, or flexible.
  • a conduit may be assembled by connecting two or more conduits. Conduits may be connected using connectors, valves, or fittings.
  • a conduit may be monolithic (e.g., a single, integrated piece).
  • the one or more conduits comprises a set of conduits connecting an outlet of the first reaction chamber to an inlet of the second reaction chamber.
  • the conduit is capable of bi-directional flow.
  • the one or more conduits comprises a first set of conduits connecting an outlet of the first reaction chamber to an inlet of the second reaction chamber, and a second set of conduits connecting an outlet of the second reaction chamber to an inlet of the first reaction chamber.
  • the systems and methods described herein further comprise at least one or more reaction chambers in addition to the first reaction chamber comprising a transferase and the second reaction chamber comprising a hydrolase.
  • Addition reaction chambers e.g., one or more reaction chambers
  • Additional enzymes include primases, additional transferases, single stranded RNA ligases, or pyrophosphatases.
  • the one or more additional reaction chambers may comprise one or more enzymes.
  • the one or more additional reaction chambers comprise a transferase and a pyrophosphatase.
  • the systems and methods described herein further comprise at least a third reaction chamber, at least a fourth reaction chamber, at least a fifth reaction chamber, a least a sixth reaction chamber, at least a seventh reaction chamber, comprising another enzyme, such as a transferase, a single stranded RNA ligase, a primase, or a pyrophosphatase.
  • another enzyme such as a transferase, a single stranded RNA ligase, a primase, or a pyrophosphatase.
  • the one or more reaction chamber comprises a second transferase. It is contemplated that some transferase variants will exhibit greater efficiency when conjugating certain nucleotides comprising a 3' blocking moiety. As such, multiple transferases may be used to conjugate different nucleotides comprising a 3' blocking moiety, depending on the pre-selected nucleotide.
  • the one or more reaction chambers can be fluidly connected to the other reaction chambers, and the flow path is selected such that the reaction chamber comprising the second transferase is utilized for elongating the oligonucleotide when appropriate.
  • the one or more reaction chambers comprises a pyrophosphatase.
  • the pyrophosphatase is an inorganic pyrophosphatase.
  • Inorganic phosphate is generated as a byproduct when the transferase conjugates the nucleotide to the oligonucleotide.
  • Inorganic phosphate can inhibit enzymes such as hydrolases used for removing the 3' blocking moiety. Thus, treating the solution comprising the oligonucleotide to break down inorganic phosphate can be used to maintain efficient reaction kinetics.
  • the pyrophosphatase is an inorganic pyrophosphatase.
  • the inorganic pyrophosphatase is fused to the transferase.
  • the one or more reaction chambers comprising the pyrophosphatase can be fluidly connected to the other reaction chambers, and the flow path is selected such that the reaction chamber comprising the pyrophosphatase is utilized after the 3' blocking moiety is removed from the elongated oligonucleotide comprising a 3' blocking moiety.
  • the one or more reaction chamber comprises a primase. Primase is a polymerase that is capable of synthesizing oligonucleotides from single nucleotides or elongating short oligonucleotides.
  • Primase is also more efficient than some hydrolases in elongating oligonucleotides shorter than 10 nucleotides in length.
  • adding primase in a reaction chamber is useful for synthesizing and/or extending oligonucleotides productive for transferase extension.
  • the one or more reaction chambers comprising the primase can be fluidly connected to the other reaction chambers, and the flow path is selected such that a reaction chamber (e.g., one of the one or more reaction chambers) comprising the primase is utilized to produce an oligonucleotide that can be elongated by the transferase in the first chamber.
  • the one or more reaction chamber comprises a single stranded RNA ligase (ssRNA ligase).
  • ssRNA ligase is an enzyme that can ligate a nucleotide donor to an oligonucleotide acceptor, or ligate an oligonucleotide donor to an oligonucleotide acceptor. Elongated nucleotides produced by the systems and methods described herein may be ligated together to produce a longer oligonucleotide.
  • the one or more reaction chambers comprising the ssRNA ligase can be fluidly connected to the other reaction chambers in the system, and the flow path is selected such that a reaction chamber (e.g., one of the one or more reaction chambers) comprising the ssRNA ligase is utilized after an oligonucleotide is elongated by the systems and methods as disclosed herein.
  • a reaction chamber e.g., one of the one or more reaction chambers
  • one or more oligonucleotides are introduced into the reaction chamber comprising the ssRNA ligase.
  • a nucleotide donor e.g., a single nucleotide donor or an oligonucleotide donor
  • a nucleotide donor is introduced into the reaction chamber comprising the ssRNA ligase along with an oligonucleotide acceptor.
  • any of the reaction chambers described herein are fluidly connected by one or more conduits.
  • the first reaction chamber and the second reaction chamber are fluidly connected by one or more conduits.
  • the third reaction chamber is fluidly connected to the first reaction chamber and the second reaction chamber by one or more conduits.
  • a conduit such as a conduit that fluidly connects two or more reaction chambers, may be used to operably link two or more reaction chambers.
  • the one or more conduits operably links two or more reaction chambers.
  • conduits include chromatography tubing of any material, including glass, plastic, rubber, and silicone.
  • the conduit may be rigid, semi-rigid, or flexible.
  • a conduit may be assembled by connecting two or more conduits. Conduits may be connected using connectors, valves, or fittings. In some aspects, a conduit may be monolithic (e.g., a single, integrated piece). In some aspects, the one or more conduits comprises a set of conduits connecting an outlet of the first reaction chamber to an inlet of the second reaction chamber. In some aspects, the conduit is capable of bidirectional flow. In some aspects, the one or more conduits comprises a first set of conduits connecting an outlet of the first reaction chamber to an inlet of the second reaction chamber, and a second set of conduits connecting an outlet of the second reaction chamber to an inlet of the first reaction chamber.
  • the enzymes for the process and methods described herein can use wild-type enzymes, engineered enzymes, and combinations of wild- type enzymes and engineered enzymes. Various combinations of such enzymes can be applied to the methods and processes as appropriate where such enzymes are used.
  • any of the enzymes described herein may be modified (e.g., comprise amino acid mutations relative to the wild-type protein) to enhance activity, enhance binding, alter activity (such as, but not limited to activity under different conditions and in the presence of various co-factors) to catalyze a reaction using modified nucleotides, increase product yield, increase protein expression, increase thermoactivity, increase thermostability, increase stability, increase substrate specificity and/or affinity, increase substrate range, increase specific activity, increase resistance to substrate and/or end-product inhibition, increase chemical stability, improve solvent stability, increase solubility, and increase inhibitor resistance or tolerance.
  • modified nucleotides e.g., comprise amino acid mutations relative to the wild-type protein
  • alter activity such as, but not limited to activity under different conditions and in the presence of various co-factors
  • any of the systems or methods using the primase, transferase, single stranded RNA ligase, hydrolase and/or pyrophosphatase can be carried out by retaining the transferase, hydrolase and/or pyrophosphatase in a chamber (e.g., a reaction chamber).
  • Retaining the enzyme can be accomplished through binding or immobilizing the primase, transferase, single stranded RNA ligase, hydrolase and/or pyrophosphatase on a substrate, such as a solid support, a filter (e.g., a porous filter), a porous substrate, a membrane (e.g., a porous membrane), or particles (e.g., beads or resin).
  • a substrate such as a solid support, a filter (e.g., a porous filter), a porous substrate, a membrane (e.g., a porous membrane), or particles (e.g., beads or resin).
  • Retaining the primase, transferase, single stranded RNA ligase, hydrolase and/or pyrophosphatase permits recycling and reuse of the primase, transferase, single stranded RNA ligase, hydrolase and/or the pyrophosphatase in each iterative cycle until a predetermined nucleotide sequence is achieved. Retaining the enzyme confers additional cost savings because the enzyme is not used once then discarded.
  • An example of a cycle (e.g., one round of elongating and deblocking the oligonucleotide) is depicted in FIG. 1.
  • the polypeptide can be entrapped in matrixes or membranes.
  • matrices include polymeric materials such as calcium-alginate, agar, k-carrageenin, polyacrylamide, and collagen.
  • the solid matrices includes, among others, activated carbon, porous ceramic, and diatomaceous earth.
  • the matrix is a particle, a membrane, or a fiber. Types of membranes include, among others, nylon, cellulose, polysulfone, or polyacrylate.
  • the enzymes described herein can be fused to a variety of polypeptide sequences, such as, by way of example and not limitation, polypeptide tags that can be used for detection, purification, immobilization on a support medium, or fusion to another protein.
  • the enzyme/polypeptide comprises an affinity tag.
  • the affinity tag is located at the N-terminus or the C-terminus of the enzyme.
  • the enzyme is fused to a polylysine, for example, for conjugation to a support medium via the amino group of the polylysine.
  • the polylysine is from 2-10 lysine units in length.
  • an affinity tag can be used as an adapter to conjugate the enzyme to a solid support (e.g., a resin) that comprises the binding moiety that binds and captures the affinity tag.
  • the affinity tag is a polypeptide tag.
  • a polypeptide tag include a histidine tag (such as a glycine -histidine, penta-histidine or a hexa-histidine tag), a GST tag, a streptavidin tag, a SUMO tag, a MBP tag, a GFP tag, or an epitope tag (such as c-Myc, Flag tag (or iterations thereof), V5, or hemagglutinin (HA)).
  • the fusion is selected or designed to preserve the activity of the enzyme.
  • the affinity tag is recognized by a resin and is used to capture, to purify, to immobilize, and/or to enrich the enzyme by using the tag to immobilize the enzyme to the resin or solid support.
  • the enzyme/polypeptide is immobilized on the surface of a support material.
  • the polypeptide is adsorbed on the support material.
  • the enzyme is immobilized on the support material by a covalent, electrostatic, or ionic bond.
  • Support materials include, among others, inorganic materials, such as alumina, silica, porous glass, ceramics, diatomaceous earth, clay, and bentonite, or organic materials, such as cellulose (CMC, DEAE- cellulose), starch, activated carbon, polyacrylamide, polystyrene, and ion-exchange resins, such as Amberlite, Sephadex, and Dowex.
  • solid supports useful for immobilizing the enzyme/polypeptide in the present disclosure include beads or resins comprising polymethacrylate with epoxide functional groups, polymethacrylate with amino epoxide functional groups, styrene/DVB copolymer or polymethacrylate with octadecyl functional groups.
  • Exemplary solid supports useful for immobilizing the enzyme/polypeptide include, but are not limited to, EnginZyme (including, EziG-1, EziG-1, and EziG-3), chitosan beads, Eupergit C, and SEPABEADs (Mitsubishi) (including EC-EP, EC-HFA/S, EXA252, EXE119 and EXE120).
  • a transferase is an enzyme that is capable of elongating an oligonucleotide by covalently ligating a nucleotide or a nucleotide analog (such as a nucleotide comprising a 3' blocking moiety) to the oligonucleotide.
  • the transferase can react a nucleotide triphosphate (NTP) comprising a 3' blocking moiety, or an analog thereof comprising a 5' phosphate analog, with the oligonucleotide to elongate the oligonucleotide.
  • NTP nucleotide triphosphate
  • the 5' phosphate analog is a 5'-(a-P- thio)phosphate moiety.
  • the NTP comprising the 3' blocking moiety or the analog thereof is a ribonucleotide. In some aspects, the NTP comprising the 3' blocking moiety or the analog thereof is a deoxyribonucleotide. In some aspects, the NTP comprising the 3' blocking moiety or the analog thereof comprises a 2' modification. In some aspects, the 2' modification of the NTP or the analog thereof is 2'-F or 2'-OMe. In some aspects, 3' blocking moiety of the NTP or the analog thereof is a phosphate moiety.
  • the transferase is a terminal deoxynucleotidyl transferase (TdT).
  • TdT is a member of the Pol X family of polymerases. Members of the diverse Pol X family are known to share certain residues, which are conserved across family members. TdT also has a high level of conservation across species for residues thought to be involved in binding divalent metal ions, ternary complex formation, and binding dNTP and DNA ligands (Dominguez et al.
  • TdTs that are contemplate for use in the systems and methods described herein may be engineered (e.g., mutated or comprise amino acid mutations) to have improved thermostability, activity at elevated temperatures, increased soluble expression or isolated protein yield, decreased by-product formation, increased affinity for NTP-3'-O-RBG and other natural or modified NTP substrates, increased affinity for oligo acceptor substrates, increased activity or specific activity on NTP-3'-O-RBG and other natural or modified NTP substrates, and/or increased activity or specific activity on various oligo acceptor substrates as compared to a wild-type TdT or other TdTs or template-independent polymerases known to those of skill in the art.
  • TdTs are capable of template-independent synthesis of oligonucleotides and polynucleotides.
  • TdTs useful in the systems and methods described herein are disclosed, for example, in PCT/US2023/076667, filed October 12, 2023, which is incorporated by reference herein in its entirety.
  • template-independent polymerases e.g., template-independent transferases
  • polyA polymerases including, but not limited to polyA polymerases, polyU polymerases and terminal urildylytransferases
  • polyU polymerases including, but not limited to polyA polymerases, polyU polymerases and terminal urildylytransferases
  • other polymerases are known to be capable of template-independent synthesis (including but not limited to reverse transcriptases, lambda, polymerase mu, and members of the X family of DNA polymerases) many of which participate in DNA repair processes be used in the systems and methods described herein.
  • the transferase comprises a single stranded RNA ligase (ssRNA ligase).
  • a hydrolase is an enzyme that is catalyzes bond cleavage with water. In some aspects, the hydrolase catalyzes the removal of the 3' blocking moiety from an oligonucleotide comprising a 3' blocking moiety.
  • Hydrolases within the scope of the invention include, but are not limited to, phosphatases (e.g., alkaline phosphatase) and include, but are not limited to, enzymes acting on ester bonds.
  • Hydrolases contemplated for the methods and systems described herein include phosphatases, such as alkaline phosphatase.
  • Alkaline phosphatase is a dimeric metalloenzyme that hydrolyzes monophosphate esters and generates inorganic phosphate as a byproduct.
  • Alkaline phosphatases are widely distributed enzymes found in both prokaryotes and eukaryotes that catalyze the hydrolysis of phosphate monoesters, with an optimal activity at alkaline pH. In mammals, alkaline phosphatases are present in the intestine (i.e., intestinal alkaline phosphatase) and placenta (i.e., placental alkaline phosphatase).
  • Phosphatases are widely used in molecular biological applications, for example for the removal of 5' -phosphate from polynucleotides or oligonucleotides for subsequent labeling with labeled ATP, reducing or preventing ligation of polynucleotides or oligonucleotides, and reducing susceptibility of polynucleotides or oligonucleotides to certain nucleases, e.g., 1 exonuclease.
  • the hydrolase is a recombinant phosphatase.
  • the recombinant phosphatase is used for cleaving phosphate monoesters or analogs thereof.
  • the hydrolase is alkaline phosphatase. In some aspects, the hydrolase is a recombinant alkaline phosphatase. In some aspects, the hydrolase is a recombinant alkaline phosphatase, or a portion thereof. Alkaline phosphatases and variants thereof are disclosed, for example, in U.S. provisional application titled “Recombinant Phosphatases” filed April 16, 2024, and in PCT/US2023/076667, filed October 12, 2023, both of which are incorporated by reference herein in their entirety.
  • the hydrolase cleaves a phosphate monoester or analog thereof.
  • the substrate phosphate monoester or analog thereof comprises a nucleotide triphosphate (NTP), a nucleotide diphosphate (NDP), nucleotide monophosphate (NMP), 3’-P-NTP, 3’-P-NDP, 3’- P-NMP, Np, NTP-D -S, NDP-D -S, NMP-D -S, 3’-P-NTP-D-S, 3’-P-NDP-f -S, 3’-P-NMP-D -S, NpS, or any combination thereof, phosphatase and a polynucleotide or oligonucleotide having a 5'- phosphate (5'-P) and/or a 3'-phosphate (3’-P).
  • the substrate phosphate monoester or analog thereof comprises a polynucleotide or oligonucleotide with a 5'-phosphate, a 3'- phosphate, a 5'-phosphorothioate (5'-S), a 3'-phosphorothioate (3'-S), or any suitable combination thereof.
  • the hydrolase can remove a 3' blocking moiety from the oligonucleotide in the solution.
  • the systems and methods disclosed herein comprises the use of a primase, which conjugates a nucleotide acceptor having a 3’ -OH group and a nucleotide donor to extend the nucleotide acceptor.
  • the nucleotide acceptor is an initiating nucleotide acceptor as disclosed herein.
  • Primase e.g., a primase-polymerase refers to an enzyme in the class of RNA polymerases that is involved in the replication of DNA by catalyzing the synthesis of short RNA molecules from ribonucleoside triphosphates.
  • a primase is a polymerase that can generate oligonucleotides de novo from an initiating nucleotide.
  • the primase reacts a nucleotide acceptor having a 3 ’-OH group with a nucleotide donor.
  • the nucleotide acceptor comprises a polynucleotide acceptor, an oligonucleotide acceptor, or an initiating nucleotide acceptor.
  • the oligonucleotide acceptor is at least 2, 3, 4, 5, 6, 8, 9, 10 nucleotides in length.
  • the polynucleotide or oligonucleotide acceptor is DNA, RNA, or a mixture of DNA and RNA.
  • the 3 ’-terminal nucleotide of the oligonucleotide acceptor is a ribonucleotide.
  • the 3 ’-terminal nucleotide of the oligonucleotide acceptor is a deoxyribonucleotide.
  • the initiating nucleotide acceptor comprises NTP, NDP, NMP, or a nucleoside.
  • the nucleotide donor comprises a blocking group or the terminating nucleotide donor comprises a 3’ -blocking group to form a 3’ -blocked extended polynucleotide or extended oligonucleotide.
  • the primase extends (e.g., elongates) the nucleotide acceptor by attachment of the nucleotide donor to the nucleotide acceptor, some aspects, the primase is a recombinant primase. In some aspects, the recombinant primase comprises a polypeptide fragment of a primase polypeptide, wherein the polypeptide fragment comprises a primase domain.
  • the recombinant primase has template-independent terminal nucleotidyl transferase activity. In some aspects, the recombinant primase has nucleotidyl transferase activity at least for a nucleotide donor dATP, ddATP, ddCTP, ddGTP, 3’ -O-methyl ATP, and/or 2’ -F-ATP.
  • a primase may be used to elongate a nucleotide (e.g., a monomeric nucleotide acceptor) or an oligonucleotide.
  • the oligonucleotide is a 2-mer, a 3-mer, a 4-mer, a 5-mer, a 6-mer, a 7-mer, a 8-mer, a 9-mer, or a 10-mer.
  • the systems and methods disclosed herein comprise a reaction chamber comprising a primase.
  • the primase is a recombinant primase.
  • the primase may be a recombinant primase, which may be cloned, produced, and isolated by known methods.
  • the primase need not need a full-length primase, as active polypeptide fragments are suitable for the methods and systems described herein.
  • the primase should include an active primes domain, and moreover should exhibit template-independent terminal nucleotidyl transferase activity. Primases and variants thereof are disclosed, for example, in U.S. provisional application titled “Recombinant Primases And Methods Of Use” filed April 16, 2024, which is incorporated by reference herein in its entirety.
  • the primase is retained in a reaction chamber. In some aspects, the primase is immobilized on a solid support. In some aspects, the primase is retained by a filter that prevents passage of the primase and allows passage of a 3' -blocked donor-acceptor oligonucleotide.
  • ssRNA ligase Single stranded RNA ligase
  • Single stranded RNA ligases can ligate short oligonucleotides to form a longer oligonucleotide in an ATP dependent manner by joining the 5'-PC>4 of a nucleotide donor to the 3'-OH of a nucleotide acceptor.
  • the nucleotide donor is a single nucleotide donor.
  • the nucleotide donor comprises a nucleoside monophosphate.
  • the nucleoside monophosphate is further modified by a 3'-blocking moiety.
  • the nucleotide donor is a pNp.
  • the single nucleotide donor comprises a 5 '-phosphate and a 3'-phosphate.
  • the nucleotide donor is an oligonucleotide donor.
  • the oligonucleotide donor comprises a 5'-phosphate and a 3'-phosphate.
  • the nucleotide acceptor is an oligonucleotide.
  • the oligonucleotide comprises a 5'-OH.
  • the oligonucleotide comprises a 5' blocking moiety.
  • the systems and methods disclosed herein comprise a reaction chamber comprising a ssRNA ligase, such as those disclosed in U.S. provisional application titled “Methods of RNA ligase mediated oligonucleotide synthesis,” filed April 16, 2024, which is incorporated by reference herein in its entirety.
  • the ssRNA ligase is retained in a reaction chamber. In some aspects, the ssRNA ligase is immobilized on a solid support. In some aspects, the ssRNA ligase is retained by a filter that prevents passage of the primase and allows passage of a 3' -blocked donor-acceptor oligonucleotide.
  • the present disclosure further provides auxiliary enzymes for template-free synthesis of an oligonucleotide.
  • auxiliary enzyme is pyrophosphatase such as inorganic pyrophosphatase, which catalyzes the conversion of conversion of inorganic pyrophosphate (PPi) to two orthophosphate ions (Pi).
  • IPP enzymes e.g., recombinant IPP enzymes
  • IPP enzymes e.g., recombinant IPP enzymes
  • Pyrophosphate is often released from reactions that utilize nucleoside triphosphates (NTP).
  • NTP nucleoside triphosphates
  • Many cellular enzymes are capable of catalyzing pyrophosphate conversion; however, the activity of most of these enzymes is not specific to a pyrophosphate substrate.
  • alkaline phosphatase will fully degrade pyrophosphate but will also hydrolyze other phosphates including the 5'-phosphates of nucleotides.
  • IPP enzymes or IPPases are known to have both high activity in the conversion of pyrophosphate to orthophosphate and relatively low activity on other phosphorylated substrates including, but not limited to, NTPs and sugar phosphates.
  • Inorganic pyrophosphate is also a known inhibitor of hydrolases such as alkaline phosphatase, and accumulation can block alkaline phosphatase activity, which interferes with the hydrolase-mediated removal of the 3' blocking moiety from the NTP.
  • the systems and methods disclosed herein comprise a reaction chamber comprising an inorganic pyrophosphatase.
  • inorganic pyrophosphatases There are three types of inorganic pyrophosphatases (Type I, Type II, Type III). Type I IPPs have been used commercially in coupled reactions to facilitate enzymatic reactions involving DNA/RNA polymerase and other reactions that produce pyrophosphate.
  • Type I IPP enzymes generally drive the hydrolysis of pyrophosphate to release heat, the H(+)-PPases couple the energy of inorganic pyrophosphate hydrolysis to proton movement across biological membranes. However, these enzymes are not readily conducive to in vitro and industrial processes.
  • Type II IPP enzymes have generally been characterized from bacterial and archaeal sources (see for example, Zyryanov et al. “Rates of Elementary Catalytic Steps for Different Metal Forms of the Family II Pyrophosphatase from Streptococcus gordonii.” Biochemistry 43(4): 1065-1074 (2004), Gajadeera et al.
  • the systems and methods described herein include the use of Type II IPPs or Type I and Type II IPP variants thereof (such as those disclosed in U.S. provisional application titled “Uses of Type II Inorganic Pyrophosphatases, filed April 16, 2024, incorporated by reference herein in its entirety).
  • any of the enzymes described herein can be provided as a fusion protein.
  • the inorganic pyrophosphatase is provided as a fusion to a transferase.
  • the enzyme is fused to a variety of polypeptide sequences, such as, by way of example and not limitation, polypeptide tags that can be used for detection and/or purification.
  • the fusion polypeptide of the enzyme comprises a glycine-histidine or histidine-tag (His-tag).
  • the fusion polypeptide of the enzyme comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA).
  • the fusion polypeptide of the enzyme comprises a GST, SUMO, Strep, MBP, or GFP tag.
  • the fusion is to the amino (N-) terminus of engineered enzyme polypeptide.
  • the fusion is to the carboxy (C-) terminus of the enzyme polypeptide.
  • the fusion polypeptide comprises a transferase and the pyrophosphatase.
  • the pyrophosphatase is fused to the C terminus of the transferase.
  • the transferase and the pyrophosphatase are connected by a polypeptide linker.
  • the systems and methods described herein further comprise one or more purification chambers.
  • the systems and methods described herein may comprise two or more different types of purification chambers, such as two or more of the types of purification chambers as described herein.
  • Purification chambers can be used to purify the oligonucleotide from reaction byproducts such as inorganic phosphate, or remove unreacted nucleotides comprising a 3' blocking moiety from the solution.
  • the purification chambers isolate and/or substantially separate the oligonucleotide from other reagents and reaction byproducts.
  • a purification chamber can be used to substantially separate and/or isolate at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of the total mole or percent amount of oligonucleotide from other reagents (e.g., enzymes) and reaction byproducts after the reservoirs.
  • Purification chambers can be vessels or chambers, and are composed of an inert material.
  • the purification chambers are columns.
  • purification chamber comprises a column.
  • the purification chamber comprises a liquid chromatography column. Liquid chromatography comprises a mobile phase and a stationary phase, and certain molecules are retained in the stationary phase based on their inherent properties while others prefer to travel with the mobile phase.
  • the purification chamber comprises a size exclusion chromatography. Size exclusion chromatography separates molecules by parameters such as molecular weight and hydrodynamic radius.
  • the purification chamber comprises an ion exchange column. Ion exchange separates molecules by inherent charge parameters when the target molecule (e.g., the oligonucleotide) is in different buffers that modify whether the molecule prefers to interact more or less with the stationary phase.
  • the purification chamber is a reverse phase column. Reverse phase chromatography relies on polar/nonpolar characteristics of the molecule for separation of the target molecule from the mixture.
  • the purification chamber is part of a tangential flow filtration system. A tangential flow filtration system involves using a filter to remove or separate contaminating particles from samples.
  • the purification chamber may be configured to remove salts, reaction byproducts and/or unreacted reagents, for example as a desalting chamber.
  • Desalting chambers are useful for separating the oligonucleotide from buffer components such as salts, small molecules (such as small molecule byproducts), impurities and/or metals. Desalting chambers are also useful for buffer exchanging the oligonucleotide, and for concentrating the oligonucleotide into a smaller volume. Desalting chambers may be used outside of the flow system, for example by collecting the product and desalting it prior to re-introducing the oligonucleotide for further reactions or collecting the product at the end of oligonucleotide synthesis.
  • Desalting chambers that are not coupled to the systems as described herein include spin cartridge filters.
  • the sample is applied to the spin cartridge filter, and centrifugal force is applied to the unit to draw the permeate through the filter.
  • the filter comprises pores of a specified molecular weight cut off, such that any of the components of the sample that are larger than the size of the cut off are significantly more retained in the retentate, and components of the sample that are smaller than the size of the cut off are flowed through with the permeate.
  • Desalting chambers may also be used as a part of the flow system, such as a tangential flow system or a cross flow filtration system.
  • Filtration techniques such as ultrafiltration, may be used, and the cartridges or conditions (for example, the flow rate to maintain appropriate transmembrane pressure, or the membrane surface required for appropriate permeate flux) are compound (e.g., product) dependent.
  • the oligonucleotide (such as an oligonucleotide of about 9 or about 10 nucleotides in length or more) is retained with the retentate, but the permeate is flowed to the waste or discarded.
  • the oligonucleotide is desalted using a desalting chamber as needed.
  • the oligonucleotide is desalted at defined intervals and/or at defined cycle numbers, is
  • the desalting chamber is a vessel, e.g., a column that is packed with resin for desalting and buffer exchange, such as G25 resin.
  • the desalting chamber is a cartridge with a membrane of a designated molecular weight cut off.
  • desalting chamber is a tangential flow device or a ultrafiltration and/or diafiltration device (e.g., cassette), such as those used in ultrafiltration/diafiltration (UF/DF).
  • the cassette comprises pores with a molecular weight cut off of 1 kilodaltons. In some aspects, the cassette comprises pores with a molecular weight cut off 3 kilodaltons.
  • the desalting chamber comprises a membrane. In some aspects, the membrane is a polyethersulfone (PES) membrane. In some aspects, the temperature of the desalting chamber is 25 °C. In some aspects, the desalting chamber is cleanable. In some aspects, the desalting chamber is cleaned in place. In some aspects, the desalting chamber is reusable. In some aspects, the desalting chamber is replaceable. In some aspects, the desalting chamber is disposable. Enzyme capture chambers
  • the systems and methods described herein further comprise one or more enzyme capture chambers.
  • Enzyme capture chambers can be used to further separate trace levels of enzymes that were not retained from the reaction reservoirs. Trace amounts of the enzymes disclosed herein may not be retained by the reaction chambers.
  • the transferase or the hydrolase may be immobilized to a solid support, but the enzyme or its active subunits may decouple during the cycle run.
  • the enzymes described herein may be further modified with an affinity tag or another purification tag to capture any free enzyme in solution.
  • a chamber such as an in-line column, may be placed at the outlet of the reaction chamber (e.g., downstream of the reaction chamber) to bind any enzyme that is flowed in solution with the oligonucleotide.
  • the chamber comprises a solid support that captures the enzyme and allows passage of the oligonucleotide.
  • the resin may be, for example, an ion exchange resin or an affinity resin that can capture the enzyme.
  • the enzyme may include a His-tag and the resin may be nickel, cobalt, or IMAC resin.
  • the enzyme capture chambers to further retain, separate, or exclude the enzyme from the solution comprising the oligonucleotide.
  • the enzyme capture chambers further remove, separate and/or isolate the enzyme such that at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% of the enzyme is retained or removed from the solution comprising the oligonucleotide.
  • the systems and methods described herein further comprise one or more reagent reservoirs.
  • Reagent reservoirs are used to hold solutions, buffers, or mixtures that provide essential reagents for template-free oligonucleotide synthesis.
  • Reagents include co-factors, buffers, and nucleotides comprising 3' blocking moiety.
  • the reagent reservoir comprises an impeller, such as an impeller as described herein.
  • the reagent reservoir is replaceable or cleanable.
  • the reagent reservoir may comprise an outlet.
  • the reagent reservoir comprises an inlet and an outlet.
  • the reagent reservoir is fluidly connected to any of the reaction chambers or product reservoirs as described herein.
  • the reagent reservoir is insulated.
  • the reagent reservoir is temperature regulated.
  • the temperature regulation can be achieved by, for example, a jacketed reservoir (e.g., a jacketed reagent reservoir such as a jacketed stir tank), or a cooling element.
  • the reagent reservoir is maintained at a temperature.
  • the temperature of the reagent reservoir is about 2 °C to 8 °C.
  • the temperature of the reagent reservoir is about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, or about 8 °C.
  • the temperature of the reagent reservoir is 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, or 8 °C. In some aspects, the temperature of the reagent reservoir is 2 °C. In some aspects, the temperature of the reagent reservoir is 3 °C. In some aspects, the temperature of the reagent reservoir is 4 °C. In some aspects, the temperature of the reagent reservoir is 5 °C. In some aspects, the temperature of the reagent reservoir is 6 °C. In some aspects, the temperature of the reagent reservoir is 7 °C. In some aspects, the temperature of the reagent reservoir is 8 °C.
  • the systems and methods described herein further comprise one or more product reservoirs.
  • the product reservoir holds the product and any byproducts that are produced from the enzymatic reaction.
  • the product reservoir comprises an inlet.
  • the product reservoir comprising an inlet and an outlet.
  • the system comprises a product reservoir comprising an inlet and an outlet, wherein the system is configured to flow the oligonucleotide in the solution from an outlet of the second reaction chamber to an inlet of the product reservoir, from an outlet of the product reservoir to an inlet of the reagent reservoir.
  • the product reservoir comprises an impeller.
  • the product reservoir is replaceable or cleanable.
  • the product reservoir is insulated.
  • the product reservoir is temperature regulated.
  • the temperature regulation can be achieved by, for example, jacketed reservoir (e.g., a jacketed product reservoir such as a jacketed stir tank), or a cooling element.
  • the product reservoir is maintained at a temperature.
  • the temperature of the product reservoir is about 2 °C to 8 °C.
  • the temperature of the product reservoir is about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, or about 8 °C.
  • the temperature of the product reservoir is 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, or 8 °C. In some aspects, the temperature of the product reservoir is 2 °C. In some aspects, the temperature of the product reservoir is 3 °C. In some aspects, the temperature of the product reservoir is 4 °C. In some aspects, the temperature of the product reservoir is 5 °C. In some aspects, the temperature of the product reservoir is 6 °C. In some aspects, the temperature of the product reservoir is 7 °C. In some aspects, the temperature of the product reservoir is 8 °C.
  • the present disclosure describes systems for template-free synthesis of an oligonucleotide. Also described herein are systems for performing the methods described herein.
  • the system comprises a first reaction chamber and a second reaction chamber.
  • the system includes one or more reaction chambers.
  • the system comprises two or more reaction chambers.
  • the system comprises three or more reaction chambers.
  • the system comprises four or more reaction chambers.
  • the system further comprises any of the reagent reservoirs, product reservoirs, and purification chambers as described herein.
  • the systems described herein includes mechanisms for limiting oxidation, such as by limiting or removing oxygen from the system.
  • Mechanisms for preventing oxidation include the use of inert gasses (such as argon or nitrogen) for inerting, purging, or sparging.
  • inert gasses such as argon or nitrogen
  • the systems described herein may be operated in an inert atmosphere or under inert atmospheric conditions.
  • system for template-free synthesis of an oligonucleotide comprising: a first reaction chamber comprising a transferase; and a second reaction chamber comprising a hydrolase: wherein the system is configured to flow an oligonucleotide in a solution from the first reaction chamber to the second reaction chamber while retaining the transferase in the first reaction chamber, and flow said oligonucleotide in the solution from the second chamber back to the first reaction chamber while retaining the hydrolase in the second reaction chamber.
  • the first reaction chamber comprises a transferase.
  • the transferase is a TdT.
  • the transferase is a member of the Pol X family.
  • the first reaction chamber further comprises a pyrophosphatase.
  • the pyrophosphatase is an inorganic pyrophosphatase.
  • the first reaction chamber comprises a transferase-pyrophosphatase fusion.
  • the second reaction chamber comprises a hydrolase.
  • the hydrolase is a phosphatase.
  • the phosphatase is an alkaline phosphatase.
  • the systems described herein may be operated in parallel.
  • multiple and identical reaction chambers may be operated in parallel (e.g., simultaneously operated).
  • the systems described herein may be operated serially.
  • the first reaction chamber or the second reaction chamber is a column.
  • the first reaction chamber comprises a fixed bed comprising the transferase immobilized on a solid support.
  • the first reaction chamber comprises a fluidized bed comprising the transferase immobilized on a solid support.
  • the fluidized bed further comprises a pyrophosphatase, such as an inorganic pyrophosphatase.
  • the first reaction chamber comprises a filter that prevents passage of the transferase and allow s passage of the oligonucleotide.
  • the first reaction chamber comprises a filter that prevents passage of the transferase and the pyrophosphatase and allows passage of the oligonucleotide.
  • the second reaction chamber comprises a fixed bed comprising the hydrolase immobilized on a solid support.
  • the second reaction chamber comprises a fluidized bed comprising the hydrolase immobilized on a solid support.
  • the second reaction chamber comprises a filter that prevents passage of the hydrolase and allows passage of the oligonucleotide.
  • the first reaction chamber or the second reaction chamber is a batch reaction chamber.
  • the batch reaction chamber comprises an impeller.
  • the first reaction chamber comprises a rotating bed reactor comprising the transferase immobilized on a solid support.
  • the second reaction chamber comprises a rotating bed reactor comprising the hydrolase immobilized on a solid support.
  • the system can further comprise a temperature regulator.
  • a temperature regulator can be column wall thermostats, a still air thermostat, forced air thermostats, passive pre-heaters, active pre-heaters, post-column liquid phase cooler, or heat exchangers.
  • the temperature regulators can also be thermal blankets or insulation sleeves, jacketed reservoirs or chambers (such as any of the reaction chambers, purification chambers, enzyme capture chambers, and/or a reagent reservoirs as described herein), water baths or ice baths. Each of these elements may also be coupled to thermometers to monitor heat or cooling.
  • the temperature regulator is configured to control the temperature of the solution in the first reaction chamber or the second reaction chamber.
  • the system comprises an in-line temperature regulator that controls the temperature of the solution in at least one of the one or more conduits.
  • the system further comprises a temperature regulator that controls the temperature of the solution in the system.
  • the temperature regulator is configured to control the temperature of the solution in the first reaction chamber or the second reaction chamber.
  • the temperature regulator is a jacketed chamber, such as a jacketed purification chamber.
  • the temperature regulator is a jacketed reaction chamber.
  • the temperature regulator is a jacketed reagent reservoir.
  • the temperature regulator is a jacketed stir tank.
  • the reagent reservoirs may be operably linked to an inert gas source, such as nitrogen or argon gas tanks to sparge the buffer and/or reagents prior to introduction into the system. Sparging with inert gasses removes oxygen from the buffers, reagents, and/or liquids in the system.
  • In-line degassing apparatuses such as an in-line vacuum pump may be optionally included in the systems as disclosed herein.
  • Degassing the buffers and/or solutions used in the system may be useful, for example, for preventing oxidation of oligonucleotides and/or the modified nucleotides used to make oligonucleotides.
  • the systems optionally include one or more in-line degassers for degassing the buffers and/or solutions.
  • the system optionally includes one or more reagent reservoirs that are operably linked to a tank comprising an inert gas (such as nitrogen or argon) such that the contents within the reservoir are sparged prior to introduction into the systems.
  • the system may include one or more in-line spargers for sparging the fluids within the system.
  • the buffers and/or solutions comprise reagents, such as reagents that are useful for template-independent oligonucleotide synthesis.
  • the system comprises one or more conduits that connects the first reaction chamber and the second reaction chamber.
  • the conduits comprise an in-line temperature regulator that controls a temperature of the solution in at least one of the one or more conduits.
  • the one or more conduits comprises a first set of conduits connecting an outlet of the first reaction chamber to an inlet of the second reaction chamber, and a second set of conduits connecting an outlet of the second reaction chamber to an inlet of the first reaction chamber.
  • the system for template free synthesis of an oligonucleotide comprises a first reaction chamber, and a second reaction chamber.
  • the system is configured to flow the oligonucleotide in a liquid phase from the first reaction chamber to the second reaction chamber. In some implementations, the system is configured to flow the oligonucleotide in the liquid phase from the second reaction chamber back to the first reaction chamber.
  • FIG. 3 illustrates an example of a system in accordance with one implementation, wherein the system comprises a first reaction chamber comprising a transferase 300 and a second reaction chamber 302 comprising a hydrolase.
  • the system can be configured such that the reservoirs can be configured to flow an oligonucleotide in solution from the first reaction chamber 300 to the second reaction chamber 302 while retaining the transferase in the first reaction chamber 300, and the oligonucleotide can be flowed from the second reaction chamber 302 to the first reaction chamber 300 while retaining the hydrolase in the second reaction chamber 302.
  • Reservoirs and reaction chambers can be, for example, batch vessels, flasks, or in line columns.
  • the reaction chambers are batch vessels that are fluidly connected by conduit 304.
  • the oligonucleotide is introduced to the system by addition to first reaction chamber 300.
  • the reservoir comprises an apparatus for mixing solutions in first reaction chamber 300.
  • Preselected nucleotide e.g., a nucleotide with a 3' blocking moiety such as a NQP
  • the solution comprising the oligonucleotide and the nucleotide comprising a 3' blocking moiety is then flowed through conduit 304 into second reaction chamber 302.
  • the first reaction chamber 300 comprises a transferase which conjugates the nucleotide comprising a 3' blocking moiety (such as, for example, an NQP) to the oligonucleotide.
  • the solution, now comprising an elongated oligonucleotide comprising a 3' blocking moiety is then flowed through conduit 304.
  • the elongated oligonucleotide comprising a 3' blocking moiety is flowed through conduit 304, and into second reaction chamber 302 comprises a hydrolase and the transferase is retained in the first reaction chamber 300.
  • Different configurations of the system can be designed to retain the transferase, such as by a membrane positioned near the outlet of first reaction chamber 300. In some implementations, the transferase is retained by a membrane at the outlet of first reaction chamber 300.
  • the transferase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin.
  • the transferase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of first reaction chamber 300.
  • the in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange.
  • Second reaction chamber 302 comprises a hydrolase, which is used to remove the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to produce an elongated oligonucleotide.
  • the elongated oligonucleotide is then flowed through conduits 304 to first reaction chamber 300.
  • the elongated oligonucleotide is flowed to first reaction chamber 300 and the hydrolase is retained in second reaction chamber 302.
  • the hydrolase is retained by a membrane positioned near the outlet of second reaction chamber 302.
  • the hydrolase is retained by a membrane at the outlet of second reaction chamber 302.
  • the hydrolase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin.
  • the hydrolase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the second reaction chamber 302.
  • the in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange.
  • transferase such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange.
  • a second nucleotide e.g., a second nucleotide, a second nucleotide with a 3' blocking moiety such as a NQP
  • the system can be reiterated to elongate the oligonucleotide such that each iteration further elongates the oligonucleotide by one nucleotide.
  • the elongated oligonucleotide upon entry into first reaction chamber 300, becomes an oligonucleotide that is introduced into the system.
  • the conduits, reservoirs and/or chambers as described above in the system may also comprise one or more ports, such as an injection port that can be used to introduce additional reagents (e.g., predetermined nucleotide substrate, buffers, reagents, and/or cofactors) at defined locations (e.g., into first reaction chamber 300 or second reaction chamber 302) in the system (e.g., into first reaction chamber 300 or second reaction chamber 302).
  • additional detectors, valves, or pumps that are configured in line with the conduits, and permit fluid connection between the chambers and/or reservoirs.
  • the system can have one or more pumps, one or more UV detectors, one or more degassers, or one or more valves integrated between the conduits in a manner that permits fluid connections between the chambers and/or reservoirs.
  • the degasser can be integrated downstream of a reagent reservoir comprising buffers and/or reagents such that the degasser is fluidly connected to the system, and the buffer and/or reagents are degassed prior to introduction into the system.
  • the degasser is an in-line degasser that is fluidly connected to the system, and the solution is degassed as it is flowed through the system.
  • the system may include a reservoir that is fluidly connected to a source of inert gas for sparging the contents of the reservoir.
  • the system may comprise one or more in-line spargers for sparging the solutions within the system.
  • the system described herein may comprise one or more purification chambers.
  • the systems described herein may comprise two purification chambers.
  • the purification chambers are different types of purification chambers.
  • the system described herein may also comprise at least one purification chamber (e.g., an enzyme capture chamber) and at least one desalting chamber that can be used to separate, purify, buffer exchange and/or concentrate the oligonucleotide.
  • Exemplary placements of a desalting chamber in the systems and methods include, but are not limited to, before or after a reaction chamber or a product reservoir.
  • the oligonucleotide is first desalted and concentrated using a desalting chamber, then flowed into a reaction chamber, where the oligonucleotide is enzymatically conjugated to a conjugation moiety.
  • the enzymatic conjugation reaction can be performed in batch.
  • the system comprises an additional reaction chamber comprising a primase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 300 and the second reaction chamber comprising the hydrolase 302).
  • the oligonucleotide is synthesized in a template independent manner comprising reacting a nucleotide acceptor having a 3’ -OH group and a nucleotide donor in presence of a primase in the additional reaction chamber that is fluidly connected to the system described herein.
  • the system comprises an additional reaction chamber comprising a ssRNA ligase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 300 and the second reaction chamber comprising the hydrolase 302).
  • the additional chamber comprises a ssRNA ligase that ligates the oligonucleotide synthesized in a template independent manner using the system described herein to another nucleotide or oligonucleotide using the ssRNA ligase contained within the additional reaction chamber that is fluidly connected to the system described herein.
  • the additional reaction chamber comprises a solid support or filter to retain the primase or ssRNA ligase in the additional reaction chamber while the oligonucleotide is flowed through the system.
  • the flow for the system is semi-automated. In some implementations, the flow for the system is automated.
  • the flow can be controlled by a device, such as a flow controller, one or more valves, one or more pumps, or one or more flow meters.
  • the flow rate can be manually or automatically adjusted.
  • the flow rate is adjusted by a device, such as a device that is connected to the system.
  • the system is connected to a device.
  • the flow rate can be adjusted by a device that is connected to the system.
  • the device is a host computer connected to a network.
  • the host computer is a client computer or a server.
  • the device can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server or handheld computing device (portable electronic device) such as a phone or tablet.
  • the device can include, for example, one or more processor(s), input devices, output devices, memory or storage devices, communication devices, and one or more analytical systems for (for example, one or more liquid chromatography systems and/or one or more mass spectrometers).
  • Software that resides in the memory or storage device may comprise, for example, an operating system and software for controlling the system (e.g., a synthesizer), such as controlling automation of the system or controlling iterative cycles of the system.
  • the system is connected to an analytical system.
  • the analytical system can be used to assess properties of the oligonucleotide that is flowed through the system.
  • Non-limiting examples include quantity of elongated oligonucleotide, sequence of the elongated oligonucleotide, mass of the elongated oligonucleotide and/or oligonucleotide comprising a 3' blocking moiety, purity of the elongated oligonucleotide, and the presence and/or quantity of any byproducts, such as the byproducts disclosed herein, generated by flowing the oligonucleotide through the system.
  • Non-limiting examples of analytical systems include UV detectors, mass spectrometers, chromatography systems.
  • the system can further comprise sensors such as pressure sensors, pH meters, conductivity meters, to monitor the conditions of the system or each individual reservoir or chamber.
  • the sensors are connected in line with the conduits such that the sensor is measuring the solution that is flowing, such as flowing in conduits, or flowing between reservoirs and/or chambers.
  • the sensors are in contact with the interior of the reservoirs or chambers of the system to monitor the conditions and/or detect changes in the solution.
  • the system may further comprise one or more valves.
  • Valves are used for multiple purposes in a liquid delivery system.
  • Non-limiting examples of valves that can be used in the system include injection or inlet valves that are used to introduce liquid sample into the system, flow path switching valves that may be used to change the flow path of the liquid, column valves for attachment of one or more columns to the system, and outlet valves for waste or sample collection.
  • the one or more valves are multiposition rotary valves.
  • the valves are single position valves.
  • the system further comprises one or more valves that selectively controls a flow pathway of the solution in the system.
  • the system comprises one or more diverter valves.
  • the system further comprises one or more valves that selectively controls a flow pathway of the solution in the system.
  • the systems disclosed herein comprise diverter valves configured to alternatively direct flow of the solution through (i) a first flow pathways comprising flow of the solution from the outlet of the first reaction chamber to an inlet of the purification chamber, and from an outlet of the purification chamber to the inlet of the first reaction chamber, or (ii) a second flow pathway comprising flow of the solution from the outlet of the first reaction chamber to the inlet of the second reaction chamber, and from an outlet of the second reaction chamber to the inlet of the first reaction chamber.
  • FIG. 4 illustrates an example of a system in accordance with one implementation.
  • the system is an example of a system that comprises a first reaction chamber comprising a transferase 400 and a second reaction chamber 402 comprising a hydrolase.
  • the system can be configured such that the reservoirs can be configured to flow an oligonucleotide in solution from the first reaction chamber 400 to the second reaction chamber 402 while retaining the transferase in the first reaction chamber 400, and the oligonucleotide can be flowed from the second reaction chamber 402 to the first reaction chamber 400 while retaining the hydrolase in the second reaction chamber 402.
  • Reservoirs and reaction chambers can be, for example, batch vessels, flasks, or in line columns.
  • the reaction chambers are vessels such as batch vessels that are fluidly connected by conduit 404, but the flow is modulated by control valve 406 .
  • FIG. 4 illustrates an example of a system in accordance with one implementation.
  • the oligonucleotide is introduced to the system by addition to a first reaction chamber 400.
  • the solution is flowed from the first reaction chamber 400 to the second reaction chamber 402 via conduit 404.
  • Flow is applied by gravity, and controlled by control valve 406.
  • the transferase is added to first reaction chamber 400.
  • the oligonucleotide comprising the 3' blocking moiety is flowed to the second chamber through control valve 406 and conduit 404.
  • the transferase is retained in the top chamber, optionally through immobilization, a membrane or a filter.
  • the hydrolase is added to second reaction chamber 402 and the elongated oligonucleotide is deblocked.
  • Product can be separated from the hydrolase by, for example, inverting the chambers and using gravity flow to re-cycle the solution into the first reaction chamber 400.
  • the hydrolase is retained in the second reaction chamber by gravity flow through a filter or a membrane, immobilized enzyme, or any combination thereof.
  • the system may further comprise one or more pumps configured to flow the solution or the liquid phase.
  • a pump is a solvent delivery system.
  • a pump can deliver a single solvent or multiple solvents.
  • the pump comprises a mixer that combines multiple solvents into a final solvent that can be delivered to the system. Pumps may be configured to deliver the solvents at a determined flow rate.
  • the one or more pumps deliver solvent at a constant flow rate.
  • the one or more pumps are configured to control a flow rate of the liquid phase.
  • the system further comprises one or more pumps configured to flow the liquid phase from the first reaction chamber to the second reaction chamber, and from the second reaction chamber to the first reaction chamber.
  • the system further comprises one or more pumps configured to flow the solution from the first reaction chamber to the second reaction chamber, and from the second reaction chamber to the first reaction chamber. In some aspects, the one or more pumps are configured to control a flow rate of the solution. In some aspects, the system further comprises one or more valves that selectively controls a flow pathway of the solution in the system.
  • FIG. 5 illustrates an example of a system in accordance with one implementation.
  • the system is an example of a system that comprises a first reaction chamber comprising a transferase 500 and a second reaction chamber 502 comprising a hydrolase.
  • the system can be configured such that the reservoirs can be configured to flow an oligonucleotide in solution from the first reaction chamber 500 to the second reaction chamber 502 while retaining the transferase in the first reaction chamber 500 and the oligonucleotide can be flowed from the second reaction chamber 502 to the first reaction chamber 500 while retaining the hydrolase in the second reaction chamber.
  • Reservoirs and reaction chambers can be, for example, batch vessels, flasks, or in line columns. In the system, the reaction chambers are batch vessels that are fluidly connected by conduits 504 and 504, and the flow is controlled by pump 508.
  • the oligonucleotide is introduced to the system by addition to first reaction chamber 500.
  • the reservoir comprises an apparatus for mixing solutions in first reaction chamber 500.
  • Preselected nucleotide e.g., a nucleotide with a 3' blocking moiety such as a NQP
  • first reaction chamber comprising a transferase 500.
  • the solution comprising the oligonucleotide and the nucleotide comprising a 3' blocking moiety is then flowed through conduit 504 into second reaction chamber comprising a hydrolase 502.
  • the movement of the solution, direction of the flow, and flow rate are parameters that can be controlled by optional components such as valves and pumps, such as pump 506.
  • the first reaction chamber 500 comprises a transferase which conjugates the nucleotide comprising a 3' blocking moiety to the oligonucleotide.
  • the solution, now comprising an elongated oligonucleotide comprising a 3' blocking moiety is then flowed through conduit 504.
  • the elongated oligonucleotide comprising a 3' blocking moiety is flowed through conduit 504, and into second reaction chamber 502 and the transferase is retained in the first reaction chamber 500.
  • the transferase is retained by a membrane positioned near the outlet of first reaction chamber 500.
  • the transferase is retained by a membrane at the outlet of first reaction chamber 500.
  • the transferase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin.
  • the transferase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the first reaction chamber 500.
  • the in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange.
  • Second reaction chamber 402 comprises a hydrolase, which is used to remove the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to produce an elongated oligonucleotide.
  • the elongated oligonucleotide is then flowed through conduits 504 to first reaction chamber 500.
  • the elongated oligonucleotide is flowed to first reaction chamber 500 and the hydrolase is retained in second reaction chamber 502.
  • the hydrolase is retained by a membrane positioned near the outlet of second reaction chamber 502.
  • the hydrolase is retained by a membrane at the outlet of second reaction chamber 502.
  • the hydrolase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin.
  • the hydrolase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the second reaction chamber 502.
  • the in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange.
  • transferase such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange.
  • Flowing the elongated oligonucleotide to first reaction chamber 500 reintroduces an oligonucleotide into the system.
  • another preselected nucleotide e.g., a second nucleotide, a second nucleotide with a 3' blocking moiety such as a NQP.
  • the system can be reiterated to elongate the oligonucleotide such that each iteration further elongates the oligonucleotide by one nucleotide.
  • the elongated oligonucleotide upon entry into first reaction chamber 500, becomes an oligonucleotide that is introduced into the system.
  • the conduits, reservoirs and/or chambers as described above in the system may comprise one or more ports, such as an injection port that can be used to introduce additional reagents (e.g., predetermined nucleotide substrate, buffers, reagents, and/or cofactors) at defined locations (e.g., into first reaction chamber 500 or second reaction chamber 502) in the system (e.g., into first reaction chamber 500 or second reaction chamber 502).
  • additional detectors, valves, or pumps that are configured in line with the conduits, and permit fluid connection between the chambers and/or reservoirs.
  • the system can have one or more pumps, one or more UV detectors, one or more degassers, or one or more valves integrated between the conduits in a manner that permits fluid connections between the chambers and/or reservoirs.
  • the degasser can be integrated downstream of a reagent reservoir comprising buffers and/or reagents such that the degasser is fluidly connected to the system, and the buffer and/or reagents are degassed prior to introduction into the system.
  • the degasser is an in-line degasser that is fluidly connected to the system, and the solution is degassed as it is flowed through the system.
  • the system may include a reservoir that is fluidly connected to a source of inert gas for sparging the contents of the reservoir.
  • the system may comprise one or more in-line spargers for sparging the solutions within the system.
  • the system described herein may comprise one or more purification chambers.
  • the systems described herein may comprise two purification chambers.
  • the purification chambers are different types of purification chambers.
  • the system described herein may also comprise at least one purification chamber (e.g., an enzyme capture chamber) and at least one desalting chamber that can be used to separate, purify, buffer exchange and/or concentrate the oligonucleotide.
  • Exemplary placements of a desalting chamber in the systems and methods include, but are not limited to, before or after a reaction chamber or a product reservoir.
  • the oligonucleotide is first desalted and concentrated using a desalting chamber, then flowed into a reaction chamber, where the oligonucleotide is enzymatically conjugated to a conjugation moiety.
  • the enzymatic conjugation reaction can be performed in batch.
  • the system comprises an additional reaction chamber comprising a primase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 500 and the second reaction chamber comprising the hydrolase 502).
  • the oligonucleotide is synthesized in a template independent manner comprising reacting a nucleotide acceptor having a 3’ -OH group and a nucleotide donor in presence of a primase in the additional reaction chamber that is fluidly connected to the system described herein.
  • the system comprises an additional reaction chamber comprising a ssRNA ligase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 500 and the second reaction chamber comprising the hydrolase 502).
  • the additional chamber comprises a ssRNA ligase that ligates the oligonucleotide synthesized in a template independent manner using the system described herein to another nucleotide or oligonucleotide using the ssRNA ligase contained within the additional reaction chamber that is fluidly connected to the system described herein.
  • the additional reaction chamber comprises a solid support or filter to retain the primase or ssRNA ligase in the additional reaction chamber while the oligonucleotide is flowed through the system.
  • the flow for the system is semi-automated. In some implementations, the flow for the system is automated.
  • the flow can be controlled by a device, such as a flow controller, one or more valves, one or more pumps, or one or more flow meters.
  • the flow rate can be manually or automatically adjusted.
  • the flow rate is adjusted by a device, such as a device that is connected to the system.
  • the system is connected to a device.
  • the flow rate can be adjusted by a device that is connected to the system.
  • the device is a host computer connected to a network.
  • the host computer is a client computer or a server.
  • the device can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server or handheld computing device (portable electronic device) such as a phone or tablet.
  • the device can include, for example, one or more processor(s), input devices, output devices, memory or storage devices, communication devices, and one or more analytical systems for (for example, one or more liquid chromatography systems and/or one or more mass spectrometers).
  • Software that resides in the memory or storage device may comprise, for example, an operating system and software for controlling the system (e.g., a synthesizer), such as controlling automation of the system or controlling iterative cycles of the system.
  • the system is connected to an analytical system.
  • the analytical system can be used to assess properties of the oligonucleotide that is flowed through the system.
  • Nonlimiting examples include quantity of elongated oligonucleotide, sequence of the elongated oligonucleotide, mass of the elongated oligonucleotide and/or oligonucleotide comprising a 3' blocking moiety, purity of the elongated oligonucleotide, and the presence and/or quantity of any byproducts, such as the byproducts disclosed herein, generated by flowing the oligonucleotide through the system.
  • Non-limiting examples of analytical systems include UV detectors, mass spectrometers, chromatography systems.
  • the system may further comprise sensors such as pressure sensors, pH meters, conductivity meters, to monitor the conditions of the system or each individual reservoir or chamber.
  • the sensors are connected in line with the conduits such that the sensor is measuring the solution that is flowing, such as flowing in conduits, or flowing between reservoirs and/or chambers.
  • the sensors are in contact with the interior of the reservoirs or chambers of the system to monitor the conditions and/or detect changes in the solution.
  • FIG. 6 illustrates an example of a system in accordance with one implementation.
  • the system is an example of a system that comprises a first reaction chamber comprising a transferase 600 and a second reaction chamber 602 comprising a hydrolase.
  • the system can be configured such that the reservoirs can be configured to flow an oligonucleotide in solution from the first reaction chamber 600 to the second reaction chamber 602 while retaining the transferase in the first reaction chamber 600, and the oligonucleotide can be flowed from the second reaction chamber 602 to the first reaction chamber 600 while retaining the hydrolase in the second reaction chamber 602 .
  • Reservoirs and reaction chambers can be, for example, batch vessels, flasks, or in line columns.
  • the reaction chambers are batch vessels that are fluidly connected by conduits 604 and 606, and the direction of the flow is controlled by control valve 608.
  • the reaction chambers are also fluidly connected by conduits 618 and 620, and the direction of the flow is controlled by control valve 616.
  • the flow can be unidirectional through each of these conduits such that the flow path for flowing the solution from first reaction chamber 600 to second reaction chamber 602 is not the same flow path for flowing the solution from second reaction chamber 602 to first reaction chamber 600.
  • the pathways can be modified using control valves 616 and 608 such that the solution can cycle through a reaction chamber (e.g., the first reaction container 600 or second reaction container 602) before advancing to the other reaction chamber.
  • the oligonucleotide is introduced to the system by addition to first reaction chamber 600.
  • the reservoir comprises an apparatus for mixing solutions in first reaction chamber 600.
  • Preselected nucleotide e.g., a nucleotide with a 3' blocking moiety such as a NQP
  • first reaction chamber comprising a transferase 600.
  • the solution comprising the oligonucleotide and the nucleotide comprising a 3' blocking moiety is then flowed through conduit 604 into second reaction chamber comprising a hydrolase 602.
  • the movement of the solution, direction of the flow, and flow rate are parameters that can be controlled by optional components such as valves and pumps, such as pump 610.
  • the first reaction chamber 600 comprises a transferase which conjugates the nucleotide comprising a 3' blocking moiety to the oligonucleotide.
  • the solution, now comprising an elongated oligonucleotide comprising a 3' blocking moiety is then flowed through conduit 604 and 606.
  • the elongated oligonucleotide comprising a 3' blocking moiety is flowed through conduit 604 and 606 through control valve 608, and into second reaction chamber 602 and the transferase is retained in the first reaction chamber 600.
  • the transferase is retained by a membrane positioned near the outlet of first reaction chamber 600.
  • the transferase is retained by a membrane at the outlet of first reaction chamber 600. In some implementations the transferase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations, the transferase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the first reaction chamber 600.
  • the in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange.
  • Second reaction chamber 602 comprises a hydrolase, which is used to remove the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to produce an elongated oligonucleotide.
  • the elongated oligonucleotide is then flowed through conduits 604 to first reaction chamber 600.
  • the elongated oligonucleotide is flowed to first reaction chamber 600 and the hydrolase is retained in second reaction chamber 602.
  • the hydrolase is retained by a membrane positioned near the outlet of second reaction chamber 602.
  • the hydrolase is retained by a membrane at the outlet of second reaction chamber 602.
  • the hydrolase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin.
  • the hydrolase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the second reaction chamber 602.
  • the in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange.
  • affinity capture e.g., nickel or cobalt resin, affinity tags, antibodies
  • a second nucleotide e.g., a second nucleotide, a second nucleotide with a 3' blocking moiety such as a NQP
  • the system can be reiterated to elongate the oligonucleotide such that each iteration further elongates the oligonucleotide by one nucleotide.
  • the elongated oligonucleotide upon entry into first reaction chamber 600, becomes an oligonucleotide that is introduced into the system.
  • the conduits, reservoirs and/or chambers as described above in the system may comprise one or more ports, such as an injection port that can be used to introduce additional reagents (e.g., predetermined nucleotide substrate, buffers, reagents, and/or cofactors) at defined locations (e.g., into first reaction chamber 600 or second reaction chamber 602) in the system (e.g., into first reaction chamber 600 or second reaction chamber 602).
  • additional detectors, valves, or pumps that are configured in line with the conduits, and permit fluid connection between the chambers and/or reservoirs.
  • the degasser can be integrated downstream of a reagent reservoir comprising buffers and/or reagents such that the degasser is fluidly connected to the system, and the buffer and/or reagents are degassed prior to introduction into the system.
  • the degasser is an in-line degasser that is fluidly connected to the system, and the solution is degassed as it is flowed through the system.
  • the system may include a reservoir that is fluidly connected to a source of inert gas for sparging the contents of the reservoir.
  • the system may comprise one or more in-line spargers for sparging the solutions within the system.
  • the system described herein may comprise one or more purification chambers.
  • the systems described herein may comprise two purification chambers.
  • the purification chambers are different types of purification chambers.
  • the system described herein may also comprise at least one purification chamber (e.g., an enzyme capture chamber) and at least one desalting chamber that can be used to separate, purify, buffer exchange and/or concentrate the oligonucleotide.
  • Exemplary placements of a desalting chamber in the systems and methods include, but are not limited to, before or after a reaction chamber or a product reservoir.
  • the oligonucleotide is first desalted and concentrated using a desalting chamber, then flowed into a reaction chamber, where the oligonucleotide is enzymatically conjugated to a conjugation moiety.
  • the enzymatic conjugation reaction can be performed in batch.
  • the system comprises an additional reaction chamber comprising a primase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 600 and the second reaction chamber comprising the hydrolase 602).
  • the oligonucleotide is synthesized in a template independent manner comprising reacting a nucleotide acceptor having a 3’ -OH group and a nucleotide donor in presence of a primase in the additional reaction chamber that is fluidly connected to the system described herein.
  • the system comprises an additional reaction chamber comprising a ssRNA ligase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 600 and the second reaction chamber comprising the hydrolase 602).
  • the additional chamber comprises a ssRNA ligase that ligates the oligonucleotide synthesized in a template independent manner using the system described herein to another nucleotide or oligonucleotide using the ssRNA ligase contained within the additional reaction chamber that is fluidly connected to the system described herein.
  • the additional reaction chamber comprises a solid support or filter to retain the primase or ssRNA ligase in the additional reaction chamber while the oligonucleotide is flowed through the system.
  • the flow for the system is semi-automated. In some implementations, the flow for the system is automated.
  • the flow can be controlled by a device, such as a flow controller, one or more valves, one or more pumps, or one or more flow meters.
  • the flow rate can be manually or automatically adjusted.
  • the flow rate is adjusted by a device, such as a device that is connected to the system.
  • the system is connected to a device.
  • the flow rate can be adjusted by a device that is connected to the system.
  • the device is a host computer connected to a network.
  • the host computer is a client computer or a server.
  • the device can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server or handheld computing device (portable electronic device) such as a phone or tablet.
  • the device can include, for example, one or more processor(s), input devices, output devices, memory or storage devices, communication devices, and one or more analytical systems for (for example, one or more liquid chromatography systems and/or one or more mass spectrometers).
  • Software that resides in the memory or storage device may comprise, for example, an operating system and software for controlling the system (e.g., a synthesizer), such as controlling automation of the system or controlling iterative cycles of the system.
  • the system is connected to an analytical system.
  • the analytical system can be used to assess properties of the oligonucleotide that is flowed through the system.
  • Non-limiting examples include quantity of elongated oligonucleotide, sequence of the elongated oligonucleotide, mass of the elongated oligonucleotide and/or oligonucleotide comprising a 3' blocking moiety, purity of the elongated oligonucleotide, and the presence and/or quantity of any byproducts, such as the byproducts disclosed herein, generated by flowing the oligonucleotide through the system.
  • Non-limiting examples of analytical systems include UV detectors, mass spectrometers, chromatography systems.
  • the system may further comprise sensors such as pressure sensors, pH meters, conductivity meters, to monitor the conditions of the system or each individual reservoir or chamber.
  • the sensors are connected in line with the conduits such that the sensor is measuring the solution that is flowing, such as flowing in conduits, or flowing between reservoirs and/or chambers.
  • the sensors are in contact with the interior of the reservoirs or chambers of the system to monitor the conditions and/or detect changes in the solution.
  • the system further comprises a third reaction chamber comprising an enzyme.
  • the system comprises a third reaction chamber that comprises a transferase.
  • the system comprises a third reaction chamber that comprises a second transferase.
  • the system further comprises a third reaction chamber comprising a second transferase, wherein the system is further configured to selectively flow said oligonucleotide in the solution from the second chamber to the third reaction chamber while retaining the hydrolase in the second reaction chamber, and flow the oligonucleotide in the solution from the third reaction chamber to the second reaction chamber while retaining the second transferase in the third reaction chamber.
  • the transferase and the second transferase are different types of transferase.
  • the system further comprises one or more valves that selectively controls a flow pathway of the solution in the system.
  • the third reaction chamber comprises a pyrophosphatase, wherein the system is configured to flow the oligonucleotide in the solution from the first reaction chamber to the third reaction chamber comprising the pyrophosphatase while retaining the transferase in the first reaction chamber, and selectively flow said oligonucleotide in the solution from the third reaction chamber comprising the pyrophosphatase to the first reaction chamber comprising the transferase or the second reaction chamber comprising the hydrolase, wherein the pyrophosphate is retained in the third reaction chamber comprising the pyrophosphatase.
  • a system for template-free synthesis of an oligonucleotide comprising: a first reaction chamber comprising a transferase; and a second reaction chamber comprising a hydrolase, wherein the system is configured to flow an oligonucleotide in a solution from the first reaction chamber to the second reaction chamber while retaining the transferase in the first reaction chamber, and flow said oligonucleotide in the solution from the second chamber back to the first reaction chamber while retaining the hydrolase in the second reaction chamber, further comprising a third reaction chamber comprising a pyrophosphatase, wherein the system is configured to flow the oligonucleotide in the solution from the first reaction chamber to the third reaction chamber comprising the pyrophosphatase while retaining the transferase in the first reaction chamber, and selectively flow said oligonucleotide in the solution from the third reaction chamber comprising the pyrophosphatase to the first reaction chamber comprising
  • the third reaction chamber comprises a ssRNA ligase wherein the system is configured to flow the oligonucleotide in the solution from the first reaction chamber to the third reaction chamber comprising the ssRNA ligase while retaining the transferase in the first reaction chamber, and selectively flow said oligonucleotide in the solution from the third reaction chamber comprising the ssRNA ligase to the first reaction chamber comprising the transferase or the second reaction chamber comprising the hydrolase, wherein the ssRNA ligase is retained in the third reaction chamber.
  • the reaction chamber comprising the ssRNA ligase comprises a filter that prevents passage of ssRNA ligase and allows passage of the ligated oligonucleotide.
  • a system for template-free synthesis of an oligonucleotide comprising: a first reaction chamber comprising a transferase; and a second reaction chamber comprising a hydrolase, wherein the system is configured to flow an oligonucleotide in a solution from the first reaction chamber to the second reaction chamber while retaining the transferase in the first reaction chamber, and flow said oligonucleotide in the solution from the second chamber back to the first reaction chamber while retaining the hydrolase in the second reaction chamber, further comprising a third reaction chamber comprising a ssRNA ligase, wherein the system is configured to flow the oligonucleotide in the solution from the first reaction chamber to the third reaction chamber comprising the ssRNA ligase while retaining the transferase in the first reaction chamber, and selectively flow said oligonucleotide in the solution from the third reaction chamber comprising the ssRNA ligase to the first reaction
  • the third reaction chamber comprises a primase wherein the system is configured to flow the oligonucleotide in the solution from the first reaction chamber to the third reaction chamber comprising the primase while retaining the transferase in the first reaction chamber, and selectively flow said oligonucleotide in the solution from the third reaction chamber comprising the primase to the first reaction chamber comprising the transferase or the second reaction chamber comprising the hydrolase, wherein the primase is retained in the third reaction chamber.
  • a system for template-free synthesis of an oligonucleotide comprising: a first reaction chamber comprising a transferase; and a second reaction chamber comprising a hydrolase, wherein the system is configured to flow an oligonucleotide in a solution from the first reaction chamber to the second reaction chamber while retaining the transferase in the first reaction chamber, and flow said oligonucleotide in the solution from the second chamber back to the first reaction chamber while retaining the hydrolase in the second reaction chamber, further comprising a third reaction chamber comprising a primase, wherein the system is configured to flow the oligonucleotide in the solution from the first reaction chamber to the third reaction chamber comprising the primase while retaining the transferase in the first reaction chamber, and selectively flow said oligonucleotide in the solution from the third reaction chamber comprising the primase to the first reaction chamber comprising the transferase or the second reaction chamber comprising
  • FIG. 7 illustrates an example of a system in accordance with one implementation.
  • the system comprises a first reaction chamber 700 comprising a transferase and a second reaction chamber 702 comprising a hydrolase that is fluidly connected by conduits 704, 706, and 708. Flow path is controlled through conduits 704, 706, and 708 by control valves 710 and 712. Second reaction chamber comprising a hydrolase 702 is fluidly connected back to first reaction chamber 700 via conduits 714, 716, and 718, and flow path is controlled by control valves 720 and 724. The flow is controlled by pump 726.
  • the system further comprises a third reaction chamber comprising a second transferase 726.
  • Third reaction chamber comprising a second transferase 726 is fluidly connected to first reaction chamber comprising a first transferase via conduits 704 and 728 through valve 710, and via conduits 730 and 718 through valve 724.
  • Third reaction chamber comprising a second transferase 726 is fluidly connected to second reaction chamber comprising a hydrolase via conduits 706 and 708 through valve 712, and via conduits 814 and 816 through valves 720 and 724.
  • Pump 726 is fluidly connected to the system using conduits 732 and 734.
  • the enzyme in the third reaction chamber is a primase.
  • the enzyme in the third reaction chamber is a ssRNA ligase.
  • the oligonucleotide is introduced to the system by addition to first reaction chamber 700.
  • the first reaction chamber comprises an apparatus for mixing solutions in first reaction chamber 700.
  • Preselected nucleotide e.g., a nucleotide with a 3' blocking moiety such as a NQP
  • first reaction chamber comprising a transferase 700.
  • the solution comprising the oligonucleotide and the nucleotide comprising a 3' blocking moiety is then flowed through conduit 704 into second reaction chamber comprising a hydrolase 702.
  • the solution is then flowed from the second reaction chamber 702 through conduits 714, 716 and 718 back to first reaction chamber comprising transferase.
  • the solution can be flowed to the third reaction chamber comprising a second transferase via conduits 714 and 716 through valves 720 and 724.
  • the movement of the solution, direction of the flow, and flow rate are parameters that can be controlled by optional components such as valves and pumps, such as control valves 720, 725, 710, 712, and pump 726.
  • the elongated oligonucleotide comprising a 3' blocking moiety is flowed through conduit 804, 806, and 808 or 806 and 808 through control valves 728 or 728 and 712 into second reaction chamber 702.
  • the system may further comprise one or more reagent reservoirs.
  • the system comprises a reagent reservoir comprising an inlet and an outlet; and wherein the system is configured to flow the oligonucleotide in the solution from an outlet of one of the reagent reservoir to the inlet of the first reaction chamber, from the outlet of the first reaction chamber to the inlet of the second reaction chamber, and from the outlet of the second reaction chamber to an inlet of the reagent reservoir.
  • the reagent reservoir comprises an impeller.
  • the reagent reservoir is replaceable or cleanable.
  • system further comprising a product reservoir comprising an inlet and an outlet, wherein the system is configured to flow the oligonucleotide in the solution from an outlet of the second reaction chamber to an inlet of the product reservoir, from an outlet of the product reservoir to an inlet of the first reaction chamber.
  • product reservoir comprises an impeller.
  • the product reservoir is replaceable or cleanable.
  • the transferase is retained by a membrane positioned near the outlet of first reaction chamber 700. In some implementations, the transferase is retained by a membrane at the outlet of first reaction chamber 700. In some implementations the transferase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations, the transferase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the first reaction chamber 700. The in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange.
  • affinity capture e.g., nickel or cobalt resin, affinity tags, antibodies
  • Second reaction chamber 702 comprises a hydrolase, which is used to remove the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to produce an elongated oligonucleotide.
  • the elongated oligonucleotide is then flowed through conduits 704 to first reaction chamber 700.
  • the elongated oligonucleotide is flowed to first reaction chamber 700 and the hydrolase is retained in second reaction chamber 702.
  • the hydrolase is retained by a membrane positioned near the outlet of second reaction chamber 702.
  • the hydrolase is retained by a membrane at the outlet of second reaction chamber 702.
  • the hydrolase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin.
  • the hydrolase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the second reaction chamber 702.
  • the in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange.
  • affinity capture e.g., nickel or cobalt resin, affinity tags, antibodies
  • the system can be reiterated to elongate the oligonucleotide such that each iteration further elongates the oligonucleotide by one nucleotide.
  • the elongated oligonucleotide upon entry into first reaction chamber 700, becomes an oligonucleotide that is introduced into the system.
  • the conduits, reservoirs and/or chambers as described above in the system may comprise one or more ports, such as an injection port that can be used to introduce additional reagents (e.g., predetermined nucleotide substrate, buffers, reagents, and/or cofactors) at defined locations (e.g., into first reaction chamber 700 or second reaction chamber 702) in the system (e.g., into first reaction chamber 800 or second reaction chamber 702).
  • additional detectors, valves, or pumps that are configured in line with the conduits, and permit fluid connection between the chambers and/or reservoirs.
  • the system can have one or more pumps, one or more UV detectors, one or more degassers, or one or more valves integrated between the conduits in a manner that permits fluid connections between the chambers and/or reservoirs.
  • the degasser can be integrated downstream of a reagent reservoir comprising buffers and/or reagents such that the degasser is fluidly connected to the system, and the buffer and/or reagents are degassed prior to introduction into the system.
  • the degasser is an in-line degasser that is fluidly connected to the system, and the solution is degassed as it is flowed through the system.
  • the system may include a reservoir that is fluidly connected to a source of inert gas for sparging the contents of the reservoir.
  • the system may comprise one or more in-line spargers for sparging the solutions within the system.
  • the system described herein may comprise one or more purification chambers.
  • the systems described herein may comprise two purification chambers.
  • the purification chambers are different types of purification chambers.
  • the system described herein may also comprise at least one purification chamber (e.g., an enzyme capture chamber) and at least one desalting chamber that can be used to separate, purify, buffer exchange and/or concentrate the oligonucleotide.
  • Exemplary placements of a desalting chamber in the systems and methods include, but are not limited to, before or after a reaction chamber or a product reservoir.
  • the oligonucleotide is first desalted and concentrated using a desalting chamber, then flowed into a reaction chamber, where the oligonucleotide is enzymatically conjugated to a conjugation moiety.
  • the enzymatic conjugation reaction can be performed in batch.
  • the system comprises an additional reaction chamber comprising a primase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 700 and the second reaction chamber comprising the hydrolase 702).
  • the oligonucleotide is synthesized in a template independent manner comprising reacting a nucleotide acceptor having a 3’ -OH group and a nucleotide donor in presence of a primase in the additional reaction chamber that is fluidly connected to the system described herein.
  • the system comprises an additional reaction chamber comprising a ssRNA ligase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 700 and the second reaction chamber comprising the hydrolase 702).
  • the additional chamber comprises a ssRNA ligase that ligates the oligonucleotide synthesized in a template independent manner using the system described herein to another nucleotide or oligonucleotide using the ssRNA ligase contained within the additional reaction chamber that is fluidly connected to the system described herein.
  • the additional reaction chamber comprises a solid support or filter to retain the primase or ssRNA ligase in the additional reaction chamber while the oligonucleotide is flowed through the system.
  • the flow for the system is semi-automated. In some implementations, the flow for the system is automated.
  • the flow can be controlled by a device, such as a flow controller, one or more valves, one or more pumps, or one or more flow meters.
  • the flow rate can be manually or automatically adjusted.
  • the flow rate is adjusted by a device, such as a device that is connected to the system.
  • the system is connected to a device.
  • the flow rate can be adjusted by a device that is connected to the system.
  • the device is a host computer connected to a network.
  • the host computer is a client computer or a server.
  • the device can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server or handheld computing device (portable electronic device) such as a phone or tablet.
  • the device can include, for example, one or more processor(s), input devices, output devices, memory or storage devices, communication devices, and one or more analytical systems for (for example, one or more liquid chromatography systems and/or one or more mass spectrometers).
  • Software that resides in the memory or storage device may comprise, for example, an operating system and software for controlling the system (e.g., a synthesizer), such as controlling automation of the system or controlling iterative cycles of the system.
  • the system is connected to an analytical system.
  • the analytical system can be used to assess properties of the oligonucleotide that is flowed through the system.
  • Nonlimiting examples include quantity of elongated oligonucleotide, sequence of the elongated oligonucleotide, mass of the elongated oligonucleotide and/or oligonucleotide comprising a 3' blocking moiety, purity of the elongated oligonucleotide, and the presence and/or quantity of any byproducts, such as the byproducts disclosed herein, generated by flowing the oligonucleotide through the system.
  • Non-limiting examples of analytical systems include UV detectors, mass spectrometers, chromatography systems.
  • the system may further comprise sensors such as pressure sensors, pH meters, conductivity meters, to monitor the conditions of the system or each individual reservoir or chamber.
  • the sensors are connected in line with the conduits such that the sensor is measuring the solution that is flowing, such as flowing in conduits, or flowing between reservoirs and/or chambers.
  • the sensors are in contact with the interior of the reservoirs or chambers of the system to monitor the conditions and/or detect changes in the solution.
  • FIG. 8 is a system in accordance with one implementation.
  • the reaction chamber comprising transferase 800 is fluidly connected to a second reaction chamber comprising hydrolase 802 by conduits 804 and 806 and valve 808.
  • Second reaction chamber is fluidly connected back to first reaction chamber comprising transferase 800 by conduits 818 and 820 by valve 816.
  • a reagent reservoir/ product reservoir 810 is also fluidly connected to the system by valves 822 and 816, and conduits 822 and 824.
  • the oligonucleotide is introduced to the system by addition to first reaction chamber 800.
  • the reservoir comprises an apparatus for mixing solutions in first reaction chamber 800.
  • Preselected nucleotide e.g., a nucleotide with a 3' blocking moiety such as a NQP
  • first reaction chamber comprising a transferase 800.
  • the solution comprising the oligonucleotide and the nucleotide comprising a 3' blocking moiety is then flowed through conduit 804 into second reaction chamber comprising a hydrolase 802.
  • the solution is then flowed from the second reaction chamber 802 through conduits 818 or 806 back to the reagent reservoir which may optionally act as a product reservoir via control valves 816 or 822, respectively.
  • the solution can be cycled through to the first reaction chamber 800.
  • the movement of the solution, direction of the flow, and flow rate are parameters that can be controlled by optional components such as valves and pumps, such as reagent reservoir 810.
  • the first reaction chamber 800 comprises a transferase which conjugates the nucleotide comprising a 3' blocking moiety to the oligonucleotide.
  • the solution, now comprising an elongated oligonucleotide comprising a 3' blocking moiety is then flowed through conduits 804 and 806.
  • the elongated oligonucleotide comprising a 3' blocking moiety is flowed through conduit 804 and 806 through control valve 808 into reagent reservoir 810 via conduit 822, mixed with new nucleotide comprising a 3' blocking moiety, and then flowed into second reaction chamber 802 using control valve 816 through conduit 818, while transferase is retained in the first reaction chamber 800.
  • FIG. 9 is further embodiment of the system of FIG.
  • the transferase is retained by a membrane positioned near the outlet of first reaction chamber 800. In some implementations, the transferase is retained by a membrane at the outlet of first reaction chamber 800. In some implementations the transferase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations, the transferase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the first reaction chamber 800.
  • the in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange.
  • Second reaction chamber 802 comprises a hydrolase, which is used to remove the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to produce an elongated oligonucleotide.
  • the elongated oligonucleotide is then flowed through conduits 804 to first reaction chamber 800.
  • the elongated oligonucleotide is flowed to first reaction chamber 800 and the hydrolase is retained in second reaction chamber 802.
  • the hydrolase is retained by a membrane positioned near the outlet of second reaction chamber 802. In some implementations, the hydrolase is retained by a membrane at the outlet of second reaction chamber 802. In some implementations the hydrolase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations, the hydrolase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the second reaction chamber 802. The in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange.
  • affinity capture e.g., nickel or cobalt resin, affinity tags, antibodies
  • Flowing the elongated oligonucleotide to first reaction chamber 800 reintroduces an oligonucleotide into the system.
  • another preselected nucleotide e.g., a second nucleotide, a second nucleotide with a 3' blocking moiety such as a NQP
  • the system can be reiterated to elongate the oligonucleotide such that each iteration further elongates the oligonucleotide by one nucleotide.
  • the elongated oligonucleotide upon entry into first reaction chamber 800, the elongated oligonucleotide becomes an oligonucleotide that is introduced into the system.
  • the conduits, reservoirs and/or chambers as described above in the system may comprise one or more ports, such as an injection port that can be used to introduce additional reagents (e.g., predetermined nucleotide substrate, buffers, reagents, and/or cofactors) at defined locations (e.g., into first reaction chamber 800 or second reaction chamber 802) in the system (e.g., into first reaction chamber 800 or second reaction chamber 802).
  • additional detectors, valves, or pumps that are configured in line with the conduits, and permit fluid connection between the chambers and/or reservoirs.
  • the system can have one or more pumps, one or more UV detectors, one or more degassers, or one or more valves integrated between the conduits in a manner that permits fluid connections between the chambers and/or reservoirs.
  • the degasser can be integrated downstream of a reagent reservoir comprising buffers and/or reagents such that the degasser is fluidly connected to the system, and the buffer and/or reagents are degassed prior to introduction into the system.
  • the degasser is an in-line degasser that is fluidly connected to the system, and the solution is degassed as it is flowed through the system.
  • the system may include a reservoir that is fluidly connected to a source of inert gas for sparging the contents of the reservoir.
  • the system may comprise one or more in-line spargers for sparging the solutions within the system.
  • the system described herein may comprise one or more purification chambers.
  • the systems described herein may comprise two purification chambers.
  • the purification chambers are different types of purification chambers.
  • the system described herein may also comprise at least one purification chamber (e.g., an enzyme capture chamber) and at least one desalting chamber that can be used to separate, purify, buffer exchange and/or concentrate the oligonucleotide.
  • Exemplary placements of a desalting chamber in the systems and methods include, but are not limited to, before or after a reaction chamber or a product reservoir.
  • the oligonucleotide is first desalted and concentrated using a desalting chamber, then flowed into a reaction chamber, where the oligonucleotide is enzymatically conjugated to a conjugation moiety.
  • the enzymatic conjugation reaction can be performed in batch.
  • the system comprises an additional reaction chamber comprising a primase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 800 and the second reaction chamber comprising the hydrolase 802).
  • the oligonucleotide is synthesized in a template independent manner comprising reacting a nucleotide acceptor having a 3 ’-OH group and a nucleotide donor in presence of a primase in the additional reaction chamber that is fluidly connected to the system described herein.
  • the system comprises an additional reaction chamber comprising a ssRNA ligase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 800 and the second reaction chamber comprising the hydrolase 802).
  • the additional chamber comprises a ssRNA ligase that ligates the oligonucleotide synthesized in a template independent manner using the system described herein to another nucleotide or oligonucleotide using the ssRNA ligase contained within the additional reaction chamber that is fluidly connected to the system described herein.
  • the additional reaction chamber comprises a solid support or filter to retain the primase or ssRNA ligase in the additional reaction chamber while the oligonucleotide is flowed through the system.
  • the flow for the system is semi-automated. In some implementations, the flow for the system is automated.
  • the flow can be controlled by a device, such as a flow controller, one or more valves, one or more pumps, or one or more flow meters.
  • the flow rate can be manually or automatically adjusted.
  • the flow rate is adjusted by a device, such as a device that is connected to the system.
  • the system is connected to a device.
  • the flow rate can be adjusted by a device that is connected to the system.
  • the device is a host computer connected to a network.
  • the host computer is a client computer or a server.
  • the device can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server or handheld computing device (portable electronic device) such as a phone or tablet.
  • the device can include, for example, one or more processor(s), input devices, output devices, memory or storage devices, communication devices, and one or more analytical systems for (for example, one or more liquid chromatography systems and/or one or more mass spectrometers).
  • Software that resides in the memory or storage device may comprise, for example, an operating system and software for controlling the system (e.g., a synthesizer), such as controlling automation of the system or controlling iterative cycles of the system.
  • the system is connected to an analytical system.
  • the analytical system can be used to assess properties of the oligonucleotide that is flowed through the system.
  • Non-limiting examples include quantity of elongated oligonucleotide, sequence of the elongated oligonucleotide, mass of the elongated oligonucleotide and/or oligonucleotide comprising a 3' blocking moiety, purity of the elongated oligonucleotide, and the presence and/or quantity of any byproducts, such as the byproducts disclosed herein, generated by flowing the oligonucleotide through the system.
  • Non-limiting examples of analytical systems include UV detectors, mass spectrometers, chromatography systems.
  • the system may further comprise sensors such as pressure sensors, pH meters, conductivity meters, to monitor the conditions of the system or each individual reservoir or chamber.
  • the sensors are connected in line with the conduits such that the sensor is measuring the solution that is flowing, such as flowing in conduits, or flowing between reservoirs and/or chambers.
  • the sensors are in contact with the interior of the reservoirs or chambers of the system to monitor the conditions and/or detect changes in the solution.
  • the system may further comprise a purification chamber.
  • the system comprises a purification chamber configured to separate unreacted nucleotides or reaction byproducts in the solution from an oligonucleotide.
  • the purification chamber comprises a column.
  • the column is a liquid chromatography column.
  • the purification chamber comprises size exclusion chromatography resin.
  • the purification chamber comprises ion-exchange resin.
  • the purification chamber comprises reverse phase resin.
  • the purification chamber is configured to separate unreacted nucleotides or reaction byproducts in the solution from an oligonucleotide.
  • the purification chamber is configured to capture incidental enzymes such as free transferase or free hydrolase that was not retained by the chamber.
  • FIG. 10 illustrates an example of a system in accordance with one implementation.
  • the system is an example of a system that comprises a first reaction chamber 1010 and a second reaction chamber 1018.
  • Reservoirs and reaction chambers can be, for example, batch vessels, flasks, or in line columns, in the system, the reaction chambers are in line columns.
  • Reagent reservoir 1000 is fluidly connected to a first reaction chamber 1010 comprising a transferase via conduits 1004 and 1008, optionally including pump 1006.
  • First reaction chamber 1010 is fluidly connected to a second reaction chamber 1018 comprising a hydrolase by conduits 1012 and 1016, optionally an AT 1014 that is in line between first reaction chamber 1010.
  • Second reaction chamber 1018 is fluidly connected to purification chamber 1026 by conduits 1020 and 1024, optionally including AT 1022.
  • Purification chamber 1028 is fluidly connected to product reservoir 1034 that comprises impeller 10106.
  • Product reservoir 1034 is fluidly connected to reagent reservoir 1000 by conduit 1040.
  • Product reservoir also is fluidly connected to conduit 10108.
  • the reagent reservoir 1000 and product reservoir are the same reservoir.
  • the oligonucleotide is introduced to the system by addition into reagent reservoir 1000.
  • the reservoir comprises an apparatus for mixing solutions in reagent reservoir 1000, such as impeller 1002.
  • Preselected nucleotide e.g., a nucleotide with a 3' blocking moiety such as a NQP
  • Impeller 1002 is used to stir the solution comprising the oligonucleotide and the nucleotide comprising a 3' blocking moiety, then the liquid is flowed through conduits 1004 and 1008 via pump 1006 into a first reaction chamber 1010.
  • the first reaction chamber 1010 comprises a transferase.
  • the transferase conjugates the nucleotide comprising a 3' blocking moiety to the oligonucleotide.
  • the solution, now comprising an elongated oligonucleotide comprising a 3' blocking moiety is then flowed through conduits 1012 and 1016, passing through AT 1014 into a second reaction chamber 1018.
  • the elongated oligonucleotide comprising a 3' blocking moiety is flowed through conduits 1012 and 1016, and into second reaction chamber 1018 and the transferase is retained in the first reaction chamber 1010.
  • the transferase is retained by a membrane positioned near the outlet of first reaction chamber 1010.
  • the transferase is retained by a membrane at the outlet of first reaction chamber 1010. In some implementations the transferase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations, the transferase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the first reaction chamber 1010.
  • the in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange.
  • Second reaction chamber 1018 comprises a hydrolase, which is used to remove the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to produce an elongated oligonucleotide.
  • the elongated oligonucleotide is flowed through conduits 1020 and 1024, and optionally via AT 1012, and into purification chamber 1026.
  • the elongated oligonucleotide is flowed to purification chamber 1026 and the hydrolase is retained in second reaction chamber 1018.
  • the hydrolase is retained by a membrane positioned near the outlet of second reaction chamber 1018.
  • the hydrolase is retained by a membrane at the outlet of second reaction chamber 1018. In some implementations the hydrolase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations, the hydrolase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the second reaction chamber 1018.
  • the in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange.
  • the elongated oligonucleotide is flowed through conduits 1020 and 1024, and into purification chamber 1026 and the hydrolase is retained in the second reaction chamber 1018.
  • the elongated oligonucleotide is optionally purified in purification chamber 1026, then flowed through conduits 1028 and 1032, and optionally via AT 1030 into product reservoir 1034.
  • Product reservoir 1034 comprises impeller 1036, which is used to stir the solution comprising the elongated oligonucleotide.
  • the elongated oligonucleotide can exit the system using conduit 1038 or can be flowed to reagent reservoir 1000 through conduit 1040.
  • Flowing the elongated oligonucleotide to reagent reservoir 1000 reintroduces an oligonucleotide into the system.
  • another preselected nucleotide e.g., a second nucleotide, a second nucleotide with a 3' blocking moiety such as a NQP
  • the system can be reiterated to elongate the oligonucleotide such that each iteration further elongates the oligonucleotide by one nucleotide.
  • the elongated oligonucleotide upon entry into reagent reservoir 1000, the elongated oligonucleotide becomes an oligonucleotide that is introduced into the system.
  • the conduits, reservoirs and/or chambers as described above in the system may also comprise one or more ports, such as an injection port that can be used to introduce additional reagents (e.g., predetermined nucleotide substrate, buffers, reagents, and/or cofactors) at defined locations (e.g., into reagent reservoir 1000, first reaction chamber 1010 or second reaction chamber 1018) in the system (e.g., into first reaction chamber 1010 or second reaction chamber 1018).
  • the system described herein comprises additional detectors, valves, or pumps that are configured in line with the conduits, and permit fluid connection between the chambers and/or reservoirs.
  • the system can have one or more pumps, one or more UV detectors, one or more degassers, or one or more valves integrated between the conduits in a manner that permits fluid connections between the chambers and/or reservoirs.
  • the degasser can be integrated downstream of a reagent reservoir comprising buffers and/or reagents such that the degasser is fluidly connected to the system, and the buffer and/or reagents are degassed prior to introduction into the system.
  • the degasser is an in-line degasser that is fluidly connected to the system, and the solution is degassed as it is flowed through the system.
  • the system may include a reservoir that is fluidly connected to a source of inert gas for sparging the contents of the reservoir.
  • the system may comprise one or more in-line spargers for sparging the solutions within the system.
  • the system described herein may comprise one or more purification chambers.
  • the systems described herein may comprise two purification chambers.
  • the purification chambers are different types of purification chambers.
  • the system described herein may also comprise at least one purification chamber (e.g., an enzyme capture chamber) and at least one desalting chamber that can be used to separate, purify, buffer exchange and/or concentrate the oligonucleotide.
  • Exemplary placements of a desalting chamber in the systems and methods include, but are not limited to, before or after a reaction chamber or a product reservoir.
  • the oligonucleotide is first desalted and concentrated using a desalting chamber, then flowed into a reaction chamber, where the oligonucleotide is enzymatically conjugated to a conjugation moiety.
  • the enzymatic conjugation reaction can be performed in batch.
  • the system comprises an additional reaction chamber comprising a primase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 1010 and the second reaction chamber comprising the hydrolase 1018).
  • the oligonucleotide is synthesized in a template independent manner comprising reacting a nucleotide acceptor having a 3’ -OH group and a nucleotide donor in presence of a primase in the additional reaction chamber that is fluidly connected to the system described herein.
  • the system comprises an additional reaction chamber comprising a ssRNA ligase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 1010 and the second reaction chamber comprising the hydrolase 1018).
  • the additional chamber comprises a ssRNA ligase that ligates the oligonucleotide synthesized in a template independent manner using the system described herein to another nucleotide or oligonucleotide using the ssRNA ligase contained within the additional reaction chamber that is fluidly connected to the system described herein.
  • the additional reaction chamber comprises a solid support or filter to retain the primase or ssRNA ligase in the additional reaction chamber while the oligonucleotide is flowed through the system. In some implementations, the flow for the system is semi-automated.
  • the device is a host computer connected to a network.
  • the host computer is a client computer or a server.
  • the device can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server or handheld computing device (portable electronic device) such as a phone or tablet.
  • the device can include, for example, one or more processor(s), input devices, output devices, memory or storage devices, communication devices, and one or more analytical systems for (for example, one or more liquid chromatography systems and/or one or more mass spectrometers).
  • Software that resides in the memory or storage device may comprise, for example, an operating system and software for controlling the system (e.g., a synthesizer), such as controlling automation of the system or controlling iterative cycles of the system.
  • the system is connected to an analytical system.
  • the analytical system can be used to assess properties of the oligonucleotide that is flowed through the system.
  • Non- limiting examples include quantity of elongated oligonucleotide, sequence of the elongated oligonucleotide, mass of the elongated oligonucleotide and/or oligonucleotide comprising a 3' blocking moiety, purity of the elongated oligonucleotide, and the presence and/or quantity of any byproducts, such as the byproducts disclosed herein, generated by flowing the oligonucleotide through the system.
  • Non-limiting examples of analytical systems include UV detectors, mass spectrometers, chromatography systems. The system may further comprise sensors such as pressure sensors, pH meters, conductivity meters, to monitor the conditions of the system or each individual reservoir or chamber.
  • the sensors are connected in line with the conduits such that the sensor is measuring the solution that is flowing, such as flowing in conduits, or flowing between reservoirs and/or chambers.
  • the sensors are in contact with the interior of the reservoirs or chambers of the system to monitor the conditions and/or detect changes in the solution.
  • FIG. 11 illustrates an example of a system in accordance with one implementation.
  • the system is an example of a system that comprises a first reaction chamber 1114 and a second reaction chamber 1122.
  • Reservoirs and reaction chambers can be, for example, batch vessels, flasks, or in line columns. In the system, the reaction chambers are in line columns.
  • Reagent reservoir 1100 comprises impeller 1102, and is fluidly connected to a first reaction chamber 1114 comprising a transferase via conduits 1104 and 1108, optionally including pump 1106.
  • First reaction chamber 1114 is fluidly connected to a second reaction chamber l l22 comprising a hydrolase by conduits 1116, 1138, 1104, 1108, 1110, 1120, valves 1112, 1120, 1118, pump 1106, and reservoir 1110.
  • Second reaction chamber 1122 is fluidly connected to purification chamber 1128 1116, 1138, 1104, 1108, 1110, 1120, 1126, 1136, valves 1112, 1120, 1118, 1132, pump 1106, and reservoir 1110.
  • Product reservoir 1034 is fluidly connected to reagent reservoir 1000 by conduit 1040.
  • the reagent reservoir 1000 and product reservoir are the same reservoir.
  • the oligonucleotide is introduced to the system by addition into reagent reservoir 1100.
  • the reservoir comprises an apparatus for mixing solutions in reagent reservoir 1100, such as impeller 1102.
  • Preselected nucleotide e.g., a nucleotide with a 3' blocking moiety such as a NQP
  • Impeller 1102 is used to stir the solution comprising the oligonucleotide and the nucleotide comprising a 3' blocking moiety, then the liquid is flowed through conduits 1104 and 1108 via pump 1106 into a diverter valve 1108.
  • the solution is flowed through conduit 1110, through two diverter valves 1112 and 1120.
  • the solution is routed to the first reaction chamber 1114 via conduit 1124, then flowed through conduit 1116 to reach another diverter valve 1118.
  • Diverter valve 1118 flows the solution back to the reagent reservoir 1100 by conduit 1138, and further rerouted through conduits 1104, 1108, and diverter valve 1118.
  • the first reaction chamber 1114 comprises a transferase.
  • the transferase conjugates the nucleotide comprising a 3' blocking moiety to the oligonucleotide.
  • the solution now comprising an elongated oligonucleotide comprising a 3' blocking moiety is then flowed through conduits 1104, 1108, and 1110 through diverter valves 1112 and 1120 into a second reaction chamber 1122 via conduit 1128.
  • Conduit 1130 routes the solution through diverter valve 1118 and conduit 1138 for return back to reagent chamber 1100.
  • This cycle between first reaction and second reaction chamber can be reiterated any number of times.
  • the oligonucleotide can optionally be purified between extension cycles or at the end of the reaction. From the reagent reservoir 1100, the solution is flowed through conduits 1104 and 1108 and 1110 by pump 1106 and through conduits 1122 and 1120 through a purification chamber 1128.
  • Conduit 1130 flows the solution to diverter valve 1132, where the purified oligonucleotide can return to reaction chamber 1100 for further extension/ deblocking using conduit 1136, or exit the system using conduits 1134 and 1138 through diverter valve 1118.
  • the transferase is retained by a membrane positioned near the outlet of first reaction chamber 1114. In some implementations, the transferase is retained by a membrane at the outlet of first reaction chamber 1114. In some implementations the transferase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin.
  • the transferase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the first reaction chamber 1114.
  • the in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange.
  • Second reaction chamber 1122 comprises a hydrolase, which is used to remove the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to produce an elongated oligonucleotide.
  • the hydrolase is retained by a membrane positioned near the outlet of second reaction chamber 1122.
  • the hydrolase is retained by a membrane at the outlet of second reaction chamber 1122. In some implementations the hydrolase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations, the hydrolase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the second reaction chamber 1122.
  • the in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange.
  • the system optionally comprises solid bed reaction chambers.
  • Flowing the elongated oligonucleotide to reagent reservoir 1100 reintroduces an oligonucleotide into the system.
  • another preselected nucleotide e.g., a second nucleotide, a second nucleotide with a 3' blocking moiety such as a NQP
  • the system can be reiterated to elongate the oligonucleotide such that each iteration further elongates the oligonucleotide by one nucleotide.
  • the elongated oligonucleotide upon entry into reagent reservoir 1100, the elongated oligonucleotide becomes an oligonucleotide that is introduced into the system.
  • the conduits, reservoirs and/or chambers as described above in the system may comprise one or more ports, such as an injection port that can be used to introduce additional reagents (e.g., predetermined nucleotide substrate, buffers, reagents, and/or cofactors) at defined locations (e.g., into reagent reservoir 1100, first reaction chamber 1114 or second reaction chamber 1122) in the system (e.g., into first reaction chamber 1114 or second reaction chamber 1122).
  • the system described herein comprises additional detectors, valves, or pumps that are configured in line with the conduits, and permit fluid connection between the chambers and/or reservoirs.
  • the system can have one or more pumps, one or more UV detectors, one or more degassers, or one or more valves integrated between the conduits in a manner that permits fluid connections between the chambers and/or reservoirs.
  • the degasser can be integrated downstream of a reagent reservoir comprising buffers and/or reagents such that the degasser is fluidly connected to the system, and the buffer and/or reagents are degassed prior to introduction into the system.
  • the degasser is an in-line degasser that is fluidly connected to the system, and the solution is degassed as it is flowed through the system.
  • the system may include a reservoir that is fluidly connected to a source of inert gas for sparging the contents of the reservoir.
  • the system may comprise one or more in-line spargers for sparging the solutions within the system.
  • the system described herein may comprise one or more purification chambers.
  • the systems described herein may comprise two purification chambers.
  • the purification chambers are different types of purification chambers.
  • the system described herein may also comprise at least one purification chamber (e.g., an enzyme capture chamber) and at least one desalting chamber that can be used to separate, purify, buffer exchange and/or concentrate the oligonucleotide.
  • Exemplary placements of a desalting chamber in the systems and methods include, but are not limited to, before or after a reaction chamber or a product reservoir.
  • the oligonucleotide is first desalted and concentrated using a desalting chamber, then flowed into a reaction chamber, where the oligonucleotide is enzymatically conjugated to a conjugation moiety.
  • the enzymatic conjugation reaction can be performed in batch.
  • the system comprises an additional reaction chamber comprising a primase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 1114 and the second reaction chamber comprising the hydrolase 1122).
  • the oligonucleotide is synthesized in a template independent manner comprising reacting a nucleotide acceptor having a 3’ -OH group and a nucleotide donor in presence of a primase in the additional reaction chamber that is fluidly connected to the system described herein.
  • the system comprises an additional reaction chamber comprising a ssRNA ligase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 1114 and the second reaction chamber comprising the hydrolase 1122).
  • the additional chamber comprises a ssRNA ligase that ligates the oligonucleotide synthesized in a template independent manner using the system described herein to another nucleotide or oligonucleotide using the ssRNA ligase contained within the additional reaction chamber that is fluidly connected to the system described herein.
  • the additional reaction chamber comprises a solid support or filter to retain the primase or ssRNA ligase in the additional reaction chamber while the oligonucleotide is flowed through the system. In some implementations, the flow for the system is semi-automated.
  • the oligonucleotide is flowed through the system using a pre-determined flow rate until it exits the system.
  • the flow for the system is automated.
  • the flow can be controlled by a device, such as a flow controller, one or more valves, one or more pumps, or one or more flow meters.
  • the flow rate can be manually or automatically adjusted.
  • the flow rate is adjusted by a device, such as a device that is connected to the system.
  • the system is connected to a device.
  • the flow rate can be adjusted by a device that is connected to the system.
  • the device is a host computer connected to a network.
  • the host computer is a client computer or a server.
  • the device can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server or handheld computing device (portable electronic device) such as a phone or tablet.
  • the device can include, for example, one or more processor(s), input devices, output devices, memory or storage devices, communication devices, and one or more analytical systems for (for example, one or more liquid chromatography systems and/or one or more mass spectrometers).
  • Software that resides in the memory or storage device may comprise, for example, an operating system and software for controlling the system (e.g., a synthesizer), such as controlling automation of the system or controlling iterative cycles of the system.
  • the system is connected to an analytical system.
  • the analytical system can be used to assess properties of the oligonucleotide that is flowed through the system.
  • Nonlimiting examples include quantity of elongated oligonucleotide, sequence of the elongated oligonucleotide, mass of the elongated oligonucleotide and/or oligonucleotide comprising a 3' blocking moiety, purity of the elongated oligonucleotide, and the presence and/or quantity of any byproducts, such as the byproducts disclosed herein, generated by flowing the oligonucleotide through the system.
  • Non-limiting examples of analytical systems include UV detectors, mass spectrometers, chromatography systems.
  • the system may further comprise sensors such as pressure sensors, pH meters, conductivity meters, to monitor the conditions of the system or each individual reservoir or chamber.
  • the sensors are connected in line with the conduits such that the sensor is measuring the solution that is flowing, such as flowing in conduits, or flowing between reservoirs and/or chambers.
  • the sensors are in contact with the interior of the reservoirs or chambers of the system to monitor the conditions and/or detect changes in the solution.
  • FIG. 12 illustrates an example of a system in accordance with one implementation.
  • the system is an example of a system that comprises a first reaction chamber 1200 and a second reaction chamber 1214.
  • Reservoirs and reaction chambers can be, for example, batch vessels, flasks, or in line columns.
  • First reaction chamber comprising a transferase is fluidly connected to second reaction chamber 1214 comprising a hydrolase.
  • First reaction chamber further comprises an impeller 1202 and second reaction chamber comprises impeller 1216.
  • the first and second reaction chambers are fluidly connected by conduits 1204, 1208, 1210, 1218, 1222, 1218, 1224, 1243 and valves 1212, 1214, and pumps 1206, 1220.
  • the oligonucleotide is introduced to the system by addition into reagent reservoir 1200.
  • the reservoir comprises an apparatus for mixing solutions in reagent reservoir 1200, such as impeller 1202.
  • Preselected nucleotide e.g., a nucleotide with a 3' blocking moiety such as a NQP
  • An optional impeller, such as Impeller 1202 is used to stir the solution comprising the oligonucleotide and the nucleotide comprising a 3' blocking moiety, then the liquid is flowed through conduits 1204 and 1208 via pump 1206 into a diverter valve 1208.
  • the solution is flowed through conduit 1210, through diverter valve 1212 into a second reaction chamber 1214, which also comprises an optional impeller 1316.
  • the first reaction chamber 1200 comprises a transferase.
  • the transferase conjugates the nucleotide comprising a 3' blocking moiety to the oligonucleotide.
  • the solution, now comprising an elongated oligonucleotide comprising a 3' blocking moiety is then flowed through conduits 1204, 1208 through pump 1206 and diverter valve 1212 into a second reaction chamber 1214.
  • Conduits 1218 and 1222 and 1232 route the solution back to the first reaction chamber 1200. This cycle between first reaction and second reaction chamber can be reiterated any number of times.
  • the oligonucleotide can optionally be purified between extension cycles or at the end of the reaction by routing the solution to the first reaction chamber as described above, flowing the solution through conduits 1204 and 1208 through pump 1206, and using diverter valve 1212 to flow the solution through purification chamber 1228.
  • Conduit 1230 is used to connect the purification chamber to diverter valve 1224, which cycles the solution to the first reaction chamber for more rounds of extension/ deblocking, purification, or collecting the product (e.g., using the first reaction chamber as a product reservoir).
  • the transferase is retained by a membrane positioned near the outlet of first reaction chamber 1 00. In some implementations, the transferase is retained by a membrane at the outlet of first reaction chamber 1200.
  • the transferase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin.
  • the transferase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the first reaction chamber 1200.
  • the in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange.
  • Second reaction chamber 1214 comprises a hydrolase, which is used to remove the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to produce an elongated oligonucleotide.
  • the hydrolase is retained by a membrane positioned near the outlet of second reaction chamber 1214. In some implementations, the hydrolase is retained by a membrane at the outlet of second reaction chamber 1214. In some implementations the hydrolase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations, the hydrolase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the second reaction chamber 1214. The in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange.
  • affinity capture e.g., nickel or cobalt resin, affinity tags, antibodies
  • Flowing the elongated oligonucleotide to reagent reservoir 1200 reintroduces an oligonucleotide into the system.
  • another preselected nucleotide e.g., a second nucleotide, a second nucleotide with a 3' blocking moiety such as a NQP
  • the system can be reiterated to elongate the oligonucleotide such that each iteration further elongates the oligonucleotide by one nucleotide.
  • the elongated oligonucleotide upon entry into reagent reservoir 1200, the elongated oligonucleotide becomes an oligonucleotide that is introduced into the system.
  • the conduits, reservoirs and/or chambers as described above in the system may comprise one or more ports, such as an injection port that can be used to introduce additional reagents (e.g., predetermined nucleotide substrate, buffers, reagents, and/or cofactors) at defined locations (e.g., into first reaction chamber 1200 or second reaction chamber 1214) in the system (e.g., into first reaction chamber 1200 or second reaction chamber 1214).
  • additional detectors, valves, or pumps that are configured in line with the conduits, and permit fluid connection between the chambers and/or reservoirs.
  • the system can have one or more pumps, one or more UV detectors, one or more degassers, or one or more valves integrated between the conduits in a manner that permits fluid connections between the chambers and/or reservoirs.
  • the degasser can be integrated downstream of a reagent reservoir comprising buffers and/or reagents such that the degasser is fluidly connected to the system, and the buffer and/or reagents are degassed prior to introduction into the system.
  • the degasser is an in-line degasser that is fluidly connected to the system, and the solution is degassed as it is flowed through the system.
  • the system may include a reservoir that is fluidly connected to a source of inert gas for sparging the contents of the reservoir.
  • the system may comprise one or more in-line spargers for sparging the solutions within the system.
  • the system described herein may comprise one or more purification chambers.
  • the systems described herein may comprise two purification chambers.
  • the purification chambers are different types of purification chambers.
  • the system described herein may also comprise at least one purification chamber (e.g., an enzyme capture chamber) and at least one desalting chamber that can be used to separate, purify, buffer exchange and/or concentrate the oligonucleotide.
  • Exemplary placements of a desalting chamber in the systems and methods include, but are not limited to, before or after a reaction chamber or a product reservoir.
  • the oligonucleotide is first desalted and concentrated using a desalting chamber, then flowed into a reaction chamber, where the oligonucleotide is enzymatically conjugated to a conjugation moiety.
  • the enzymatic conjugation reaction can be performed in batch.
  • the system comprises an additional reaction chamber comprising a primase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 1200 and the second reaction chamber comprising the hydrolase 1214).
  • the oligonucleotide is synthesized in a template independent manner comprising reacting a nucleotide acceptor having a 3’ -OH group and a nucleotide donor in presence of a primase in the additional reaction chamber that is fluidly connected to the system described herein.
  • the system comprises an additional reaction chamber comprising a ssRNA ligase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 1200 and the second reaction chamber comprising the hydrolase 1214).
  • the additional chamber comprises a ssRNA ligase that ligates the oligonucleotide synthesized in a template independent manner using the system described herein to another nucleotide or oligonucleotide using the ssRNA ligase contained within the additional reaction chamber that is fluidly connected to the system described herein.
  • the additional reaction chamber comprises a solid support or filter to retain the primase or ssRNA ligase in the additional reaction chamber while the oligonucleotide is flowed through the system. In some implementations, the flow for the system is semi-automated.
  • the oligonucleotide is flowed through the system using a pre-determined flow rate until it exits the system.
  • the flow for the system is automated.
  • the flow can be controlled by a device, such as a flow controller, one or more valves, one or more pumps, or one or more flow meters.
  • the flow rate can be manually or automatically adjusted.
  • the flow rate is adjusted by a device, such as a device that is connected to the system.
  • the system is connected to a device.
  • the flow rate can be adjusted by a device that is connected to the system.
  • the device is a host computer connected to a network.
  • the host computer is a client computer or a server.
  • the device can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server or handheld computing device (portable electronic device) such as a phone or tablet.
  • the device can include, for example, one or more processor(s), input devices, output devices, memory or storage devices, communication devices, and one or more analytical systems for (for example, one or more liquid chromatography systems and/or one or more mass spectrometers).
  • Software that resides in the memory or storage device may comprise, for example, an operating system and software for controlling the system (e.g., a synthesizer), such as controlling automation of the system or controlling iterative cycles of the system.
  • the system is connected to an analytical system.
  • the analytical system can be used to assess properties of the oligonucleotide that is flowed through the system.
  • Non-limiting examples include quantity of elongated oligonucleotide, sequence of the elongated oligonucleotide, mass of the elongated oligonucleotide and/or oligonucleotide comprising a 3' blocking moiety, purity of the elongated oligonucleotide, and the presence and/or quantity of any byproducts, such as the byproducts disclosed herein, generated by flowing the oligonucleotide through the system.
  • Non-limiting examples of analytical systems include UV detectors, mass spectrometers, chromatography systems.
  • the system may further comprise sensors such as pressure sensors, pH meters, conductivity meters, to monitor the conditions of the system or each individual reservoir or chamber.
  • the sensors are connected in line with the conduits such that the sensor is measuring the solution that is flowing, such as flowing in conduits, or flowing between reservoirs and/or chambers.
  • the sensors are in contact with the interior of the reservoirs or chambers of the system to monitor the conditions and/or detect changes in the solution.
  • the system is configured for template-free synthesis of an oligonucleotide comprising: a reagent reservoir; a first column configured to substantially separate a transferase from an oligonucleotide comprising a 3' blocking moiety; and a second column configured to substantially separate a hydrolase from an oligonucleotide without the 3' blocking moiety; wherein the system is configured to (i) flow a solution comprising an oligonucleotide comprising the 3' blocking moiety and a transferase from the reagent reservoir to the first column, (ii) substantially separate the oligonucleotide comprising the 3' blocking moiety from the transferase in the first column, (iii) flow the solution comprising the oligonucleotide comprising the 3' blocking moiety from the first column to the reagent reservoir, (iv) flow the solution comprising an oligonucleotide without the 3' blocking moiety and a hydro
  • the system comprises a plurality of conduits that connects the first column, the second column, and the reagent reservoir.
  • the system comprises a purification chamber configured to separate unreacted nucleotides or reaction byproducts in the solution from the oligonucleotide comprising the 3' blocking moiety or the oligonucleotide without the 3' blocking moiety.
  • FIG. 13 illustrates an example of a system in accordance with one implementation.
  • the system is an example of a system that comprises a first reaction chamber comprising a transferase 1300 that acts as a second reaction chamber that comprises a hydrolase, wherein the chamber optionally comprises impeller 1302.
  • the chamber comprises one enzyme at a time.
  • Reservoirs and reaction chambers can be, for example, batch vessels, flasks, or in line columns.
  • the reaction chamber is fluidly connected to a series of purification chambers 1320, 1326, 1332 to remove transferase, hydrolase, and/or to purify the oligonucleotide.
  • reaction chamber 1300 These purification chambers are fluidly connected to reaction chamber 1300 by conduits 1304, 1308, 1316, 1318, 1324, 1330, 1322, 1328, 1340, 1334, 1344, and through valves 1319, 1314, 1338, 1336, 1342.
  • the flow is controlled by pump
  • the system is also fluidly connected to reagent chamber 1318.
  • reaction chamber 1300 comprising a transferase is fluidly connected to a purification column that removes transferase 1320 via conduits 1304, 1308, 1352, 1318, 1322, 1342, 1344, and 1346.
  • the flow is driven by pump 1306 and the flow path is regulated by diverter valves 1310, 1314, 1336, and 1344.
  • the transferase can optionally be TdT.
  • Reaction chamber 1300 comprising a hydrolase is fluidly connected to a purification column that removes hydrolase 1326 via conduits 1304, 1308, 1352, 1324, 1328, 1342, 1344, and 1346.
  • the flow is driven by pump 1306 and the flow path is regulated by diverter valves 1310, 1314, 1336, and 1344.
  • the hydrolase can optionally be alkaline phosphatase.
  • Reaction chamber 1300 can also act as a product reservoir, and is fluidly connected to a purification column that purifies the oligonucleotide 1332 via conduits 1304, 1308, 1352, 1354, 1330, 1334, 1340, 1342, 1344, and 1346.
  • the flow is driven by pump 1306 and the flow path is regulated by diverter valves 1310, 1314, 1336, 1338, and 1344.
  • the system comprises a reagent reservoir 1318, which optionally comprises wash buffer(s).
  • Reagent reservoir 1318 is replaceable or cleanable.
  • Reagent reservoir 1318 is fluidly connected to the three purification chambers 1320, 1326, and 1332 via conduits 1316, 1352, and 1324.
  • the flow path is regulated by valve 1314.
  • the oligonucleotide is introduced to the system by addition into a first reaction chamber 1300.
  • the first reaction chamber and the second reaction chamber are the same chamber.
  • the chamber comprises one enzyme at a time.
  • the solution is mixed, optionally using impeller 1302, then flowed through conduits 1304 and 1308 using pump 1306 to diverter valve 1 10.
  • the solution is flowed through diverter valve 1324 through conduit 1318 into a column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety 1320.
  • the reservoir comprises an apparatus for mixing solutions in reagent reservoir 1300, such as impeller 1302.
  • Preselected nucleotide e.g., a nucleotide with a 3' blocking moiety such as a NQP
  • NQP a nucleotide with a 3' blocking moiety
  • Impeller 1302 is used to stir the solution comprising the oligonucleotide and the nucleotide comprising a 3' blocking moiety, then the liquid is flowed through conduits 1304 and 1308 via pump 1306 into a diverter valve 1308. From the diverter valve, the solution is flowed through conduit 1310, through diverter valve 1312 into a second reaction chamber 1314, which also comprises an optional impeller 1316.
  • the first reaction chamber 1300 comprises a transferase. The transferase conjugates the nucleotide comprising a 3' blocking moiety to the oligonucleotide.
  • the solution, now comprising an elongated oligonucleotide comprising a 3' blocking moiety is then flowed through conduits 1304, 1308 through pump 1306 and diverter valves 1310 and 1314 to remove the hydrolase via column 1326.
  • the solution is flowed through conduits 1328, 1344 and 1346 to return to first reaction chamber 1300.
  • This cycle between first reaction and second reaction chamber can be reiterated any number of times.
  • the oligonucleotide can optionally be purified between extension cycles or at the end of the reaction by routing the solution to the first reaction chamber as described above, flowing the solution through conduits 1304 and 1308 through pump 1306, and using diverter valves 1310 and 1314 to flow the solution through purification chamber 1432.
  • Conduit 1434 is used to connect the purification chamber to diverter valve 1338, which cycles the solution to the first reaction chamber 1300 through valves 1436, and conduits 1340, 1342, 1344, and 1346 for more rounds of extension/ deblocking, purification, or collecting the product (e.g., using the first reaction chamber as a product reservoir).
  • the reagent may leave the system post purification via diverter valve 1438 and conduit 1448.
  • the transferase is retained by a membrane positioned near the outlet of first reaction chamber 1400. In some implementations, the transferase is retained by a membrane at the outlet of first reaction chamber 1400.
  • the transferase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin.
  • the transferase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the first reaction chamber 1300.
  • the in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange.
  • Second reaction chamber 1314 comprises a hydrolase, which is used to remove the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to produce an elongated oligonucleotide.
  • the hydrolase is retained by a membrane positioned near the outlet of second reaction chamber 1314. In some implementations, the hydrolase is retained by a membrane at the outlet of second reaction chamber 1314. In some implementations the hydrolase is retained by immobilizing the transferase on a matrix or support, such as a bead or resin. In some implementations, the hydrolase that is incidentally released may be captured by an in-line column positioned downstream near the outlet of the second reaction chamber 1314. The in-line column comprises resin or beads that can be used for capturing transferase, such as but not limited to affinity capture (e.g., nickel or cobalt resin, affinity tags, antibodies) and ion exchange.
  • affinity capture e.g., nickel or cobalt resin, affinity tags, antibodies
  • Flowing the elongated oligonucleotide to reagent reservoir 1300 reintroduces an oligonucleotide into the system.
  • another preselected nucleotide e.g., a second nucleotide, a second nucleotide with a 3' blocking moiety such as a NQP
  • the system can be reiterated to elongate the oligonucleotide such that each iteration further elongates the oligonucleotide by one nucleotide.
  • the elongated oligonucleotide upon entry into reagent reservoir 1300, the elongated oligonucleotide becomes an oligonucleotide that is introduced into the system.
  • the conduits, reservoirs and/or chambers as described above in the system may comprise one or more ports, such as an injection port that can be used to introduce additional reagents (e.g., predetermined nucleotide substrate, buffers, reagents, and/or cofactors) at defined locations (e.g., into first reaction chamber 1300) in the system (e.g., into first reaction chamber 1300).
  • additional reagents e.g., predetermined nucleotide substrate, buffers, reagents, and/or cofactors
  • the system described herein comprises additional detectors, valves, or pumps that are configured in line with the conduits, and permit fluid connection between the chambers and/or reservoirs.
  • the system can have one or more pumps, one or more UV detectors, one or more degassers, or one or more valves integrated between the conduits in a manner that permits fluid connections between the chambers and/or reservoirs.
  • the degasser can be integrated downstream of a reagent reservoir comprising buffers and/or reagents such that the degasser is fluidly connected to the system, and the buffer and/or reagents are degassed prior to introduction into the system.
  • the degasser is an in-line degasser that is fluidly connected to the system, and the solution is degassed as it is flowed through the system.
  • the system may include a reservoir that is fluidly connected to a source of inert gas for sparging the contents of the reservoir.
  • the system may comprise one or more in-line spargers for sparging the solutions within the system.
  • the system described herein may comprise one or more purification chambers.
  • the systems described herein may comprise two purification chambers.
  • the purification chambers are different types of purification chambers.
  • the system described herein may also comprise at least one purification chamber (e.g., an enzyme capture chamber) and at least one desalting chamber that can be used to separate, purify, buffer exchange and/or concentrate the oligonucleotide.
  • Exemplary placements of a desalting chamber in the systems and methods include, but are not limited to, before or after a reaction chamber or a product reservoir.
  • the oligonucleotide is first desalted and concentrated using a desalting chamber, then flowed into a reaction chamber, where the oligonucleotide is enzymatically conjugated to a conjugation moiety.
  • the enzymatic conjugation reaction can be performed in batch.
  • the system comprises an additional reaction chamber comprising a primase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 1300 and the second reaction chamber comprising the hydrolase 1314).
  • the oligonucleotide is synthesized in a template independent manner comprising reacting a nucleotide acceptor having a 3 ’-OH group and a nucleotide donor in presence of a primase in the additional reaction chamber that is fluidly connected to the system described herein.
  • the system comprises an additional reaction chamber comprising a ssRNA ligase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 1300 and the second reaction chamber comprising the hydrolase 1314).
  • the additional chamber comprises a ssRNA ligase that ligates the oligonucleotide synthesized in a template independent manner using the system described herein to another nucleotide or oligonucleotide using the ssRNA ligase contained within the additional reaction chamber that is fluidly connected to the system described herein.
  • the additional reaction chamber comprises a solid support or filter to retain the primase or ssRNA ligase in the additional reaction chamber while the oligonucleotide is flowed through the system. In some implementations, the flow for the system is semi-automated.
  • the oligonucleotide is flowed through the system using a predetermined flow rate until it exits the system.
  • the flow for the system is automated.
  • the flow can be controlled by a device, such as a flow controller, one or more valves, one or more pumps, or one or more flow meters.
  • the flow rate can be manually or automatically adjusted.
  • the flow rate is adjusted by a device, such as a device that is connected to the system.
  • the system is connected to a device.
  • the flow rate can be adjusted by a device that is connected to the system.
  • the device is a host computer connected to a network.
  • the host computer is a client computer or a server.
  • the device can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server or handheld computing device (portable electronic device) such as a phone or tablet.
  • the device can include, for example, one or more processor(s), input devices, output devices, memory or storage devices, communication devices, and one or more analytical systems for (for example, one or more liquid chromatography systems and/or one or more mass spectrometers).
  • Software that resides in the memory or storage device may comprise, for example, an operating system and software for controlling the system (e.g., a synthesizer), such as controlling automation of the system or controlling iterative cycles of the system.
  • the system is connected to an analytical system.
  • the analytical system can be used to assess properties of the oligonucleotide that is flowed through the system.
  • Non-limiting examples include quantity of elongated oligonucleotide, sequence of the elongated oligonucleotide, mass of the elongated oligonucleotide and/or oligonucleotide comprising a 3' blocking moiety, purity of the elongated oligonucleotide, and the presence and/or quantity of any byproducts, such as the byproducts disclosed herein, generated by flowing the oligonucleotide through the system.
  • Non-limiting examples of analytical systems include UV detectors, mass spectrometers, chromatography systems.
  • the system may further comprise sensors such as pressure sensors, pH meters, conductivity meters, to monitor the conditions of the system or each individual reservoir or chamber.
  • the sensors are connected in line with the conduits such that the sensor is measuring the solution that is flowing, such as flowing in conduits, or flowing between reservoirs and/or chambers.
  • the sensors are in contact with the interior of the reservoirs or chambers of the system to monitor the conditions and/or detect changes in the solution.
  • FIG. 14 illustrates an example of a system in accordance with one implementation.
  • the system is an example of a system that comprises a first reaction chamber comprising a transferase 1400 and a second reaction chamber that comprises a hydrolase 1402.
  • Reservoirs and reaction chambers can be, for example, batch vessels, flasks, or in line columns.
  • First reaction chamber 1400 is fluidly connected to reagent reservoir/ product reservoir 1404 by conduits 1414 and 1418.
  • Conduits 1414 and 1418 are fluidly connected to a series of in-line meters or detectors 1416.
  • Optional in-line meters or detectors include flow meter 1442, UV-Vis detector 1444, conductivity meter 1446 and pH meter 1448.
  • Reagent reservoir/ product reservoir 1404 comprises optional impeller 1438 and is fluidly connected to first reaction chamber 1400 by conduits 1406, 1410, and 1452 and comprise in-line pump 1408 to flow the fluid through the conduits, and an in-line degasser 1412 and a pressure meter 1440.
  • Reagent reservoir/ product reservoir 1404 is also fluidly connected to a second reaction chamber comprising a hydrolase 1402 via conduit 1440 which optionally comprises pump 1422, inline degasser 1424 and pressure meter 1454.
  • the second reaction chamber comprising a hydrolase 1402 is fluidly connected to a reagent reservoir/ product reservoir 1434 comprising optional impeller 1436 via conduits 1450 and 1432.
  • Optional meters such as a UV-Vis detector 1428 and pressure meter 1430 are included between conduits 1450 and 1432.
  • the oligonucleotide is introduced to the system by addition into reagent reservoir/product reservoir 1404, which comprises optional impeller 1438.
  • the oligonucleotide is flowed through conduits 1406, 1410, and 1452 by pump 1408, passing through degasser 1412 and pressure meter 1440, and enters first reaction chamber comprising a transferase 1400.
  • the oligonucleotide is elongated at 40 °C to produce an oligonucleotide comprising a 3' blocking moiety, then travels through conduit 1414 and 1418, passing through optional detectors such as flow meter 1442, UV-Vis detector 1444, conductivity meter 1446, and pH meter 1448 into a reagent reservoir/ product reservoir 1404 comprising optional impeller 1438.
  • reagent reservoir/ product reservoir 1404 is a jacketed stir tank which is optionally maintained between 2 °C to 8 °C, such as at 4 °C.
  • the oligonucleotide comprising the 3' blocking moiety is then flowed through conduit 1420 by pump 1422 through an in-line degassing unit 1424 and pressure meter 1454, through conduit 1426 and into second reaction chamber comprising a hydrolase 1402.
  • second reaction chamber comprising a hydrolase 1402 is a packed bed reactor comprising the hydrolase such as alkaline phosphatase.
  • the oligonucleotide comprising a 3' blocking moiety is deblocked at 50 °C to produce an elongated oligonucleotide, which travels through conduits 1450 and 1432, through UV-Vis detector 1428 and pressure meter 1430, to reagent reservoir/ product reservoir 1434 comprising optional impeller 1436.
  • reagent reservoir/product reservoir 1434 is a jacketed stir tank which can be optionally maintained between 2 °C to 8 °C, such as at 4 °C.
  • the elongated nucleotide can then be collected from reagent reservoir/ product reservoir 1434 and reintroduced into reagent reservoir product reservoir 1404, which re-enters the elongated nucleotide into the system.
  • the system as described in FIG. 14 can be reiterated such that each iteration further elongates the oligonucleotide by one nucleotide until an oligonucleotide of a specified sequence is made.
  • Flowing the elongated oligonucleotide to reagent reservoir 1404 reintroduces an oligonucleotide into the system.
  • another preselected nucleotide e.g., a second nucleotide, a second nucleotide with a 3' blocking moiety such as a NQP
  • the system can be reiterated to elongate the oligonucleotide such that each iteration further elongates the oligonucleotide by one nucleotide.
  • the elongated oligonucleotide upon entry into reagent reservoir 1404, the elongated oligonucleotide becomes an oligonucleotide that is introduced into the system.
  • the conduits, reservoirs and/or chambers as described above in the system may comprise one or more ports, such as an injection port that can be used to introduce additional reagents (e.g., predetermined nucleotide substrate, buffers, reagents, and/or cofactors) at defined locations (e.g., into first reaction chamber 1400) in the system (e.g., into first reaction chamber 1400).
  • additional reagents e.g., predetermined nucleotide substrate, buffers, reagents, and/or cofactors
  • the system described herein comprises additional detectors, valves, or pumps that are configured in line with the conduits, and permit fluid connection between the chambers and/or reservoirs.
  • the system can have one or more pumps, one or more UV detectors, one or more degassers, or one or more valves integrated between the conduits in a manner that permits fluid connections between the chambers and/or reservoirs.
  • the degasser can be integrated downstream of a reagent reservoir comprising buffers and/or reagents such that the degasser is fluidly connected to the system, and the buffer and/or reagents are degassed prior to introduction into the system.
  • the degasser is an in-line degasser that is fluidly connected to the system, and the solution is degassed as it is flowed through the system.
  • the system may include a reservoir that is fluidly connected to a source of inert gas for sparging the contents of the reservoir.
  • the system may comprise one or more in-line spargers for sparging the solutions within the system.
  • the system described herein may comprise one or more purification chambers.
  • the systems described herein may comprise two purification chambers.
  • the purification chambers are different types of purification chambers.
  • the system described herein may also comprise at least one purification chamber (e.g., an enzyme capture chamber) and at least one desalting chamber that can be used to separate, purify, buffer exchange and/or concentrate the oligonucleotide.
  • Exemplary placements of a desalting chamber in the systems and methods include, but are not limited to, before or after a reaction chamber or a product reservoir.
  • the oligonucleotide is first desalted and concentrated using a desalting chamber, then flowed into a reaction chamber, where the oligonucleotide is enzymatically conjugated to a conjugation moiety.
  • the enzymatic conjugation reaction can be performed in batch.
  • the system comprises an additional reaction chamber comprising a primase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 1400 and the second reaction chamber comprising the hydrolase 1402).
  • the oligonucleotide is synthesized in a template independent manner comprising reacting a nucleotide acceptor having a 3'-OH group and a nucleotide donor in presence of a primase in the additional reaction chamber that is fluidly connected to the system described herein.
  • the system comprises an additional reaction chamber comprising a ssRNA ligase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 1400 and the second reaction chamber comprising the hydrolase 1402).
  • the additional chamber comprises a ssRNA ligase that ligates the oligonucleotide synthesized in a template independent manner using the system described herein to another nucleotide or oligonucleotide using the ssRNA ligase contained within the additional reaction chamber that is fluidly connected to the system described herein.
  • the additional reaction chamber comprises a solid support or filter to retain the primase or ssRNA ligase in the additional reaction chamber while the oligonucleotide is flowed through the system. In some implementations, the flow for the system is semi-automated.
  • the oligonucleotide is flowed through the system using a predetermined flow rate until it exits the system.
  • the flow for the system is automated.
  • the flow can be controlled by a device, such as a flow controller, one or more valves, one or more pumps, or one or more flow meters.
  • the flow rate can be manually or automatically adjusted.
  • the flow rate is adjusted by a device, such as a device that is connected to the system.
  • the system is connected to a device.
  • the flow rate can be adjusted by a device that is connected to the system.
  • the device is a host computer connected to a network.
  • the host computer is a client computer or a server.
  • the device can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server or handheld computing device (portable electronic device) such as a phone or tablet.
  • the device can include, for example, one or more processor(s), input devices, output devices, memory or storage devices, communication devices, and one or more analytical systems for (for example, one or more liquid chromatography systems and/or one or more mass spectrometers).
  • Software that resides in the memory or storage device may comprise, for example, an operating system and software for controlling the system (e.g., a synthesizer), such as controlling automation of the system or controlling iterative cycles of the system.
  • the system is connected to an analytical system.
  • the analytical system can be used to assess properties of the oligonucleotide that is flowed through the system.
  • Non-limiting examples include quantity of elongated oligonucleotide, sequence of the elongated oligonucleotide, mass of the elongated oligonucleotide and/or oligonucleotide comprising a 3' blocking moiety, purity of the elongated oligonucleotide, and the presence and/or quantity of any byproducts, such as the byproducts disclosed herein, generated by flowing the oligonucleotide through the system.
  • Non-limiting examples of analytical systems include UV detectors, mass spectrometers, chromatography systems.
  • the system may further comprise sensors such as pressure sensors, pH meters, conductivity meters, to monitor the conditions of the system or each individual reservoir or chamber.
  • the sensors are connected in line with the conduits such that the sensor is measuring the solution that is flowing, such as flowing in conduits, or flowing between reservoirs and/or chambers.
  • the sensors are in contact with the interior of the reservoirs or chambers of the system to monitor the conditions and/or detect changes in the solution.
  • FIG. 15 illustrates an example of a system in accordance with one implementation.
  • the system is an example of a system that comprises a first reaction chamber comprising a transferase 1500 and optional impeller 1508 and a second reaction chamber that comprises a hydrolase 1502 and optional impeller 1524.
  • Reservoirs and reaction chambers can be, for example, batch vessels, flasks, or in line columns.
  • First reaction chamber 1500 is fluidly connected to reagent reservoir/ product reservoir 1506 by conduit 1504.
  • Reagent reservoir/ product reservoir 1506 is fluidly connected to synthesizer 1512 which comprises pH meter 1514, conductivity meter 1516, and UV-Vis detector 1518.
  • Synthesizer 1512 is fluidly connected to first reaction chamber comprising a transferase 1500 via conduit 1540, to desalting chamber 1530 via conduit 1528, and to second reaction chamber comprising a hydrolase 1502 via conduit 1520. Synthesizer 1512 is also fluidly connected to reagent reservoirs comprising NQPs and buffers, such as reagent reservoirs 1534 and 1536. Second reaction chamber comprising a hydrolase 1502 is fluidly connected to reagent reservoir/product reservoir 1538 comprising impeller 1524, which is fluidly connected to the synthesizer via conduit 1526. Desalting chamber 1530 is fluidly connected to reagent reservoir/ product reservoir 1538 via conduit 1524.
  • the oligonucleotide is introduced to the system by addition into reagent reservoir/product reservoir 1506 or synthesizer 1512.
  • the system comprises a first reaction chamber comprising a transferase 1500 and a second reaction chamber comprising a hydrolase 1502.
  • the oligonucleotide is elongated at 40 °C to produce an oligonucleotide comprising a 3' blocking moiety, then travels through conduit 1504 into a reagent reservoir/ product reservoir 1506, optionally comprising impeller 1508.
  • Reagent reservoir/ product reservoir 1506 can be a jacketed stir tank, which is optionally maintained between 2 °C to 8 °C.
  • the oligonucleotide comprising the 3' blocking moiety is then flowed through bidirectional conduit 1510 and travels through synthesizer 1512 comprising optional meters that can be used to monitor the conditions of the system.
  • the meters or detectors include pH meter 1514, conductivity meter 1516 and UV-Vis detector 1518.
  • the oligonucleotide comprising the 3' blocking moiety is flowed into second reaction chamber comprising a hydrolase 1502.
  • the oligonucleotide comprising the 3' blocking moiety is deblocked to produce an elongated oligonucleotide, then the elongated oligonucleotide is flowed via conduit 1522 into a second reagent chamber/ product reservoir 1538 comprising impeller 1524.
  • the elongated oligonucleotide is flowed via conduit 1526 to synthesizer 1512, where it flows through pH meter 1514, UV-Vis detector 1518, and conductivity meter 1516.
  • the elongated oligonucleotide travels through conduit 1528 to desalting chamber 1530, where the oligonucleotide is separated, and buffer exchanged.
  • the elongated oligonucleotide travels via conduit 1532 into reagent reservoir/ product reservoir 1526.
  • the elongated oligonucleotide can either be collected from reagent reservoir/ product reservoir 1526, or rerouted through conduit 1520, through synthesizer 1512 and its meters to re-enter the first reaction chamber comprising a transferase 1500 via conduit 1540, where the elongated oligonucleotide is further elongated by the transferase to produce a oligonucleotide comprises a 3' blocking moiety.
  • the oligonucleotide comprising a 3' blocking moiety can be flowed through the system as described in FIG.
  • each iteration further elongates the oligonucleotide by one nucleotide until an oligonucleotide of a specified sequence is made.
  • the elongated oligonucleotide upon entry into reagent reservoir 1506, becomes an oligonucleotide that is introduced into the system.
  • the conduits, reservoirs and/or chambers as described above in the system may comprise one or more ports, such as an injection port that can be used to introduce additional reagents (e.g., predetermined nucleotide substrate, buffers, reagents, and/or cofactors) at defined locations (e.g., into first reaction chamber 1500) in the system (e.g., into first reaction chamber 1500).
  • additional reagents e.g., predetermined nucleotide substrate, buffers, reagents, and/or cofactors
  • the system described herein comprises additional detectors, valves, or pumps that are configured in line with the conduits, and permit fluid connection between the chambers and/or reservoirs.
  • the system can have one or more pumps, one or more UV detectors, one or more degassers, or one or more valves integrated between the conduits in a manner that permits fluid connections between the chambers and/or reservoirs.
  • the degasser can be integrated downstream of a reagent reservoir comprising buffers and/or reagents such that the degasser is fluidly connected to the system, and the buffer and/or reagents are degassed prior to introduction into the system.
  • the degasser is an in-line degasser that is fluidly connected to the system, and the solution is degassed as it is flowed through the system.
  • the system may include a reservoir that is fluidly connected to a source of inert gas for sparging the contents of the reservoir.
  • the system may comprise one or more in-line spargers for sparging the solutions within the system.
  • the system described herein may comprise one or more purification chambers.
  • the systems described herein may comprise two purification chambers.
  • the purification chambers are different types of purification chambers.
  • the system described herein may also comprise at least one purification chamber (e.g., an enzyme capture chamber) and at least one desalting chamber that can be used to separate, purify, buffer exchange and/or concentrate the oligonucleotide.
  • Exemplary placements of a desalting chamber in the systems and methods include, but are not limited to, before or after a reaction chamber or a product reservoir.
  • the oligonucleotide is first desalted and concentrated using a desalting chamber, then flowed into a reaction chamber, where the oligonucleotide is enzymatically conjugated to a conjugation moiety.
  • the enzymatic conjugation reaction can be performed in batch.
  • the system comprises an additional reaction chamber comprising a primase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 1500 and the second reaction chamber comprising the hydrolase 1502).
  • the oligonucleotide is synthesized in a template independent manner comprising reacting a nucleotide acceptor having a 3'-OH group and a nucleotide donor in presence of a primase in the additional reaction chamber that is fluidly connected to the system described herein.
  • the system comprises an additional reaction chamber comprising a ssRNA ligase that is fluidly connected to the system disclosed herein (e.g., the first reaction chamber comprising the transferase 1500 and the second reaction chamber comprising the hydrolase 1502).
  • the additional chamber comprises a ssRNA ligase that ligates the oligonucleotide synthesized in a template independent manner using the system described herein to another nucleotide or oligonucleotide using the ssRNA ligase contained within the additional reaction chamber that is fluidly connected to the system described herein.
  • the additional reaction chamber comprises a solid support or filter to retain the primase or ssRNA ligase in the additional reaction chamber while the oligonucleotide is flowed through the system. In some implementations, the flow for the system is semi-automated.
  • the oligonucleotide is flowed through the system using a pre-determined flow rate until it exits the system.
  • the flow for the system is automated.
  • the flow can be controlled by a device, such as a flow controller, one or more valves, one or more pumps, or one or more flow meters.
  • the flow rate can be manually or automatically adjusted.
  • the flow rate is adjusted by a device, such as a device that is connected to the system.
  • the system is connected to a device.
  • the flow rate can be adjusted by a device that is connected to the system.
  • the device is a host computer connected to a network.
  • the host computer is a client computer or a server.
  • the device can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server or handheld computing device (portable electronic device) such as a phone or tablet.
  • the device can include, for example, one or more processor(s), input devices, output devices, memory or storage devices, communication devices, and one or more analytical systems for (for example, one or more liquid chromatography systems and/or one or more mass spectrometers).
  • Software that resides in the memory or storage device may comprise, for example, an operating system and software for controlling the system (e.g., a synthesizer), such as controlling automation of the system or controlling iterative cycles of the system.
  • the system is connected to an analytical system.
  • the analytical system can be used to assess properties of the oligonucleotide that is flowed through the system.
  • Non-limiting examples include quantity of elongated oligonucleotide, sequence of the elongated oligonucleotide, mass of the elongated oligonucleotide and/or oligonucleotide comprising a 3' blocking moiety, purity of the elongated oligonucleotide, and the presence and/or quantity of any byproducts, such as the byproducts disclosed herein, generated by flowing the oligonucleotide through the system.
  • Non-limiting examples of analytical systems include UV detectors, mass spectrometers, chromatography systems. The system may further comprise sensors such as pressure sensors, pH meters, conductivity meters, to monitor the conditions of the system or each individual reservoir or chamber.
  • the sensors are connected in line with the conduits such that the sensor is measuring the solution that is flowing, such as flowing in conduits, or flowing between reservoirs and/or chambers.
  • the sensors are in contact with the interior of the reservoirs or chambers of the system to monitor the conditions and/or detect changes in the solution.
  • a fraction of the bound or immobilized enzyme may be stripped or may disengage from the solid support, or pass through the filter or membrane along with the elongated oligonucleotide comprising a 3' blocking moiety, or the elongated oligonucleotide.
  • the system may further comprise one or more in-line purification chambers to be used for removing any contaminating or trace levels of enzyme that exits the reaction chambers along with the nucleic acid (e.g., an oligonucleotide, an elongated oligonucleotide, or an elongated oligonucleotide comprising a 3' blocking moiety).
  • the in-line chamber is downstream of the first reaction chamber.
  • the in-line chamber is immediately downstream of the first reaction chamber.
  • the in-line chamber is downstream of the second reaction chamber.
  • the in-line chamber is immediately downstream of the second reaction chamber.
  • the in-line chamber is a purification chamber.
  • the in-line chamber comprises affinity resin, ion exchange resin, or other resin or matrices that are known to separate molecules and macromolecules in solution based on their inherent proteins.
  • the in-line chamber comprises a resin that captures the histidine tag.
  • the resin is nickel, cobalt, or immobilized metal affinity chromatography (IMAC).
  • the system may further comprise additional filters or membranes to be used for removing any contaminating or trace levels of enzyme that exits the reaction chambers along with the nucleic acid (e.g., an oligonucleotide, an elongated oligonucleotide, or an elongated oligonucleotide comprising a 3' blocking moiety).
  • the present disclosure provides for methods of template-free synthesis of an oligonucleotide, and methods that use the system described herein for template-free synthesis of an oligonucleotide.
  • any of the methods disclosed herein for template-free synthesis of an oligonucleotide can be carried out under a range of suitable reaction conditions, including but not limited to ranges of substrates, temperature, pH, solvent system, substrate loading, polypeptide loading, cofactor loading, and reaction time. Additional techniques known to one skilled in the art may be carried out to supplement the reaction conditions. These can include taking measures to stabilize or prevent inactivation of the enzyme, reduce product inhibition, shift reaction equilibrium to formation of the desired product.
  • the methods disclosed herein synthesizes the oligonucleotide to be elongated by the transferase using a primase contained in an additional reaction chamber that is fluidly connected to the system (e.g., first reaction chamber and the second reaction chamber).
  • the oligonucleotide is synthesized in a template independent manner comprising reacting a nucleotide acceptor having a 3 ’-OH group and a nucleotide donor in presence of a primase in the additional reaction chamber that is fluidly connected to the system described herein.
  • the primase is retained in the additional reaction chamber by a solid support or filter to retain the primase in the additional reaction chamber while the oligonucleotide is flowed through the system.
  • the methods provided herein further comprises ligating an oligonucleotide acceptor with a nucleotide donor (e.g., a single nucleotide donor or an oligonucleotide donor) using a ssRNA ligase.
  • a nucleotide donor e.g., a single nucleotide donor or an oligonucleotide donor
  • the ssRNA ligase that ligates the oligonucleotide synthesized in a template independent manner using the methods and systems system described herein to another nucleotide or oligonucleotide using the ssRNA ligase contained within the additional reaction chamber that is fluidly connected to the system described herein.
  • the ssRNA ligase is retained in the additional reaction chamber by a solid support or filter to retain the ssRNA ligase in the additional reaction chamber while the oligonucleotide is flowed through the system.
  • the methods provided herein include synthesizing oligonucleotides without one or more impurities (e.g., unintentionally modified oligonucleotides).
  • Oligonucleotide synthesis using enzyme mediated elongation and deblocking has advantages of avoiding impurities generated by phosphoramidite chemistry oligonucleotide synthesis, such as product related impurities that arise from chemically mediated oligonucleotide synthesis.
  • the downstream application oligonucleotides are commercial or therapeutic in nature, impurities that compromise oligonucleotide quality (such as, but not limited to oligonucleotide degradation or oligonucleotides that are unintentionally modified such as chemically modified, side-reaction modified, or degraded oligonucleotides) are undesirable.
  • the one or more impurities may be impurities that are generated during chemical synthesis of oligonucleotides.
  • the impurity is a depurinated oligonucleotide. Depurination is the loss of a purine base from a nucleotide backbone, resulting in an unintended abasic nucleotide.
  • the impurity is a depyrimidated oligonucleotide.
  • Depyrimidation is the loss of a pyrimidine base from a nucleotide backbone, resulting in an unintended abasic nucleotide.
  • the impurity is an oligonucleotide comprising a c-Net modification. cNet modified oligonucleotides comprise a N3-(2- cyanothethy)thymine in place of an intended thymine nucleotide.
  • the impurity is an oligonucleotide comprising a methyl group (e.g., a methylated oligonucleotide).
  • the methyl group is on a cytosine such that the impurity is an oligonucleotide comprising a methylcytosine in place of an intended cytosine nucleotide.
  • impurity is an oligonucleotide comprising an acetyl or isobutyryldiaminopurine (IBU) in place of an intended guanine.
  • the composition comprising the oligonucleotide made with any of the systems or methods described herein is substantially free of one or more impurities.
  • the composition comprising the oligonucleotide made by the methods or systems described herein is substantially free of the identified impurity, such that the composition comprises about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, about 0.5% or less, about 0.25% or less or about 0.1% or less of the identified impurity.
  • the composition comprising the oligonucleotide made by the methods described herein is substantially free of the identified impurity, such that the composition comprises 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.25% or less or 0.1% or less of the identified impurity. In some aspects, the composition comprising the oligonucleotide made by the methods described herein is substantially free of the identified impurity, such that the composition comprises about less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.25%, or less than 0.1% of the identified impurity. In some aspects, the identified impurity is not detectable in the composition.
  • the oligonucleotide is substantially free of depurination or depyrimidination impurities. In some aspects, the oligonucleotide is substantially free of N-3- cyanoethylthymine (CNET) impurities. In some aspects, the oligonucleotide is substantially free of N(2)-actyl-2,6-diaminopurine and/or an isobutyryl diaminopurine impurities. In some aspects, the oligonucleotide is substantially free of methylcytosine.
  • CNET cyanoethylthymine
  • the composition comprising the oligonucleotide synthesized using the method described herein have less than 30%, 25%, 20%, 15%, 10%, 5%, or 2% or less of phosphorothioate internucleoside linkages converted to the corresponding phosphodiester linkages.
  • the composition comprising the oligonucleotide synthesized using the methods described herein is substantially free of Class IV impurities, as described in Capaldi et al., Nucl Acid Ther., 2017, 27(6):309, incorporated by reference herein.
  • the composition of the synthesized oligonucleotide has no detectable Class IV impurities under standard assay conditions.
  • Class IV impurities contain “structural elements not found in the parent oligonucleotide or in naturally occurring nucleic acids, and impurities of unknown structure.” (see, e.g., Table 1 of Capaldi et al., incorporated by reference herein).
  • template free synthesis of an oligonucleotide can be carried out at about 30 °C to about 95 °C.
  • the reaction temperature is about 35 °C to about 90 °C, about 40 °C to about 85 °C, about 45 °C to about 80 °C, about 50 °C to about 75 °C, or about 55 °C to about 70 °C.
  • the suitable reaction conditions for the reaction with the recombinant primase comprises a temperature of about 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, or 95 °C.
  • the suitable reaction conditions for the reaction with the transferase comprises a temperature of about 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, or 95 °C.
  • the suitable reaction conditions for the reaction with the transferase comprises a temperature of 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, or 95 °C. In some embodiments, the suitable reaction conditions for the reaction with the transferase comprises a temperature of 40 °C.
  • the suitable reaction conditions for the reaction with the inorganic pyrophosphatase comprises a temperature of about 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, or 95 °C.
  • the suitable reaction conditions for the reaction with the inorganic pyrophosphatase comprises a temperature of 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, or 95 °C.
  • the suitable reaction conditions for the reaction with the inorganic pyrophosphatase comprises a temperature of 40 °C.
  • the suitable reaction conditions for the reaction with the hydrolase comprises a temperature of about 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, or 95 °C.
  • the suitable reaction conditions for the reaction with the hydrolase comprises a temperature of 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, or 95 °C.
  • the suitable reaction conditions for the reaction with the hydrolase comprises a temperature of 50 °C.
  • the methods of template free synthesis described herein can be carried out using buffers and/or solutions are degassed before or during operation of the systems as described herein.
  • the methods of template free synthesis described herein can be carried out using buffers and/or solutions are sparged before or during operation of the systems as described herein. Sparging adds a desired gas to the buffer, which saturates the buffer with the desired gas at the exclusion of an undesired gas.
  • the buffers and/or solutions may include reagents, such as reducing agents or antioxidants, that prevent or minimize oxidation.
  • the antioxidant is used at a final concentration of about 2 mM to about 10 mM, about 1 mM to about 5 mM, about 0.5 mM to about 8 mM, about 30-80 mM, about 10-90 mM, about 40-70 mM, or about 0-100 mM.
  • the antioxidant is used at a final concentration of about 0 mM, about 0.5 mM, about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM.
  • the antioxidant is used at 0 mM, 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM.
  • the antioxidants or reducing agents can be at a higher concentration, such as from about 100 mM to about 500 mM, from about 150 to 450 mM, from about 200 mM to about 400 mM. In some embodiments, the antioxidant or reducing agent can be about 100 mM, 150 mM, 200 mM, 300 mM, 400 mM, or 500 mM. In some aspects, the antioxidant is used at a final concentration of 5 mM. In some aspects, the antioxidant is ascorbic acid, citric acid, formic acid, sodium thiosulfate, metabisulfite, 2,6-dimethoxyphenol, or catalase. In some aspects, the antioxidant is ascorbic acid, citric acid, formic acid, sodium thiosulfate, metabisulfite, or 2,6-dimethoxyphenol. In some aspects, the antioxidant is sodium thiosulfate.
  • the antioxidant is sodium thiosulfate.
  • sodium thiosulfate is used at a final concentration of about 2 mM to about 10 mM, about 1 mM to about 5 mM, about 0.5 mM to about 8 mM, about 30-80 mM, about 10-90 mM, about 40-70 mM, or about 0-100 mM.
  • sodium thiosulfate is used at a final concentration of about 0 mM, about 0.5 mM, about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM.
  • sodium thiosulfate is used at 0 mM, 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM. In some aspects, sodium thiosulfate is used at a final concentration of 5 mM.
  • the antioxidant is catalase.
  • An example of use of the catalase in the methods as described herein is for the disproportionation of hydrogen peroxide.
  • the methods comprise about 3000 units-7000 units, about 2000-6000 units, or about 1000-8000 units of catalase.
  • the methods comprise about 3000 units, about 4000 units, about 5000 units, about 6000 units, or about 7000 units of catalase.
  • the methods comprise 3000 units, about 4000 units, about 5000 units, about 6000 units, or about 7000 units of catalase.
  • the methods comprise 5600 units (0.7 mg/ml) of catalase.
  • Template free synthesis can also be carried out at a pH suitable for the activities of any of the enzymes as disclosed herein.
  • the pH can be about 6-9, pH of about 6.5- 8.5 , or pH of about 7-8.
  • the pH of the reaction can be about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, or about 9.
  • the pH of the reaction is 6, 6.5, 7, 7.5, 8, 8.5, or 9.
  • the residence time of each cycle is concentration-dependent on the oligonucleotide.
  • Residence time is the amount of time a fluid is inside of a vessel, such as the chambers or reservoirs as described herein.
  • the extension residence time is concentration dependent.
  • the extension residence time is about 0.1, about 0.2, about 0.3 hours.
  • the extension residence time is 0.1, 0.2, or 0.3 hours.
  • the extension residence time is 0.1 hours.
  • the deblocking residence time is about 0.1, about 0.2, about 0.3 hours.
  • the deblocking residence time is 0.1, 0.2, or 0.3 hours.
  • the deblocking residence time is 0.1 hours.
  • the reaction chamber is a column. In some aspects, the column comprises 3 grams of resin. In some aspects, the first reaction chamber comprising a hydrolase comprises 3 grams of resin. In some aspects, the second reaction chamber comprising a transferase comprises 3 grams of resin.
  • any of the above-described methods for template free-synthesis of an oligonucleotide can further comprise one or more steps selected from: extraction; isolation; or purification of the oligonucleotide.
  • Methods, techniques, and protocols for extracting, isolating, purifying, and/or crystallizing the product from biocatalytic reaction mixtures produced by the above disclosed processes are known to the ordinary artisan and/or accessed through routine experimentation.
  • oligonucleotide elongating an oligonucleotide in solution by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide using a transferase to make an elongated oligonucleotide comprising the 3' blocking moiety; separating the elongated oligonucleotide comprising the 3' blocking moiety from the transferase; removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the 3' blocking moiety, the elongated oligonucleotide comprising the 3' blocking moiety being in solution when the 3' blocking moiety is removed.
  • the nucleotide comprising the 3' blocking moiety is a preselected type of nucleotide.
  • the 3' blocking moiety is removed from the elongated oligonucleotide comprising the 3' blocking moiety using a hydrolase, such as a phosphatase.
  • a hydrolase such as a phosphatase.
  • the hydrolase is an alkaline phosphatase.
  • the method further comprises separating the elongated oligonucleotide without the 3' blocking moiety from the hydrolase. Separating (e.g., substantially separating) can be done by immobilizing the hydrolase on a solid support, or using a filter or membrane to entrap or prevent passage of the hydrolase while the oligonucleotide flows through the pores.
  • the filter or membrane has a MWCO of a pre-determined value.
  • the method further comprises repeating the method for one or more cycles to further elongate the elongated oligonucleotide without the 3' blocking moiety.
  • the one or more cycles can be repeated at least once, at least twice, at least three, at least four, at least five, at least six times.
  • the method further comprises using a plurality of predetermined nucleotides are used to elongate the oligonucleotide, thereby generating an elongated oligonucleotide having a predetermined sequence, wherein each cycle after a first cycle attaches a single nucleotide to a previously elongated oligonucleotide without the 3' blocking moiety.
  • An oligonucleotide of a predetermined sequence may be generated by single addition of each pre-determined nucleotide to achieve said sequence.
  • the method further comprises using at least two different types of transferases in in separate cycles. Some transferases are better optimized to conjugate or ligate certain modified nucleotides to the oligonucleotide, due to reasons such as steric considerations that influence binding and/or efficient ligation.
  • the method comprises elongating the oligonucleotide in a first reaction chamber.
  • the method comprises removing the 3' blocking moiety in a second reaction chamber.
  • Also provided herein are methods of template-free synthesis of an oligonucleotide comprising: in a first reaction chamber, elongating an oligonucleotide in solution by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide using a transferase to make an elongated oligonucleotide comprising the 3' blocking moiety; flowing the elongated oligonucleotide comprising the 3' blocking moiety in the solution to a second reaction chamber, wherein the transferase is retained in the first reaction chamber; in the second reaction chamber, removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the 3' blocking moiety, the elongated oligonucleotide comprising the 3' blocking moiety being in solution when the 3' blocking moiety is removed; and flowing the
  • the nucleotide comprising the 3' blocking moiety is a preselected type of nucleotide.
  • the 3' blocking moiety is removed from the elongated oligonucleotide comprising the 3' blocking moiety using a hydrolase, such as a phosphatase.
  • a hydrolase such as a phosphatase.
  • the hydrolase is an alkaline phosphatase.
  • the method further comprises separating the elongated oligonucleotide without the 3' blocking moiety from the hydrolase. Separating (e.g., substantially separating) can be done by immobilizing the hydrolase on a solid support, or using a filter or membrane to entrap or prevent passage of the hydrolase while the oligonucleotide flows through the pores.
  • the filter or membrane has a MWCO of a pre-determined value.
  • the method further comprises repeating the method for one or more cycles to further elongate the elongated oligonucleotide without the 3' blocking moiety.
  • the one or more cycles can be repeated at least once, at least twice, at least three, at least four, at least five, at least six times.
  • the method further comprises using a plurality of predetermined nucleotides are used to elongate the oligonucleotide, thereby generating an elongated oligonucleotide having a predetermined sequence, wherein each cycle after a first cycle attaches a single nucleotide to a previously elongated oligonucleotide without the 3' blocking moiety.
  • An oligonucleotide of a predetermined sequence may be generated by single addition of each pre-determined nucleotide to achieve said sequence.
  • the method further comprises using at least two different types of transferases in in separate cycles. Some transferases are better optimized to conjugate or ligate certain modified nucleotides to the oligonucleotide, due to reasons such as steric considerations that influence binding and/or efficient ligation.
  • the method comprises elongating the oligonucleotide in a first reaction chamber.
  • the method comprises removing the 3' blocking moiety in a second reaction chamber.
  • conduits such as a conduit that fluidly connects two reaction chambers, may be used to operably link two reaction chambers.
  • the one or more conduits operably links two or more reaction chambers.
  • conduits include chromatography tubing of any material, including glass, plastic, rubber, and silicone.
  • the conduit may be rigid, semi-rigid, or flexible.
  • a conduit may be assembled by connecting two or more conduits. Conduits may be connected using connectors, valves, or fittings. In some aspects, a conduit may be monolithic (e.g., a single, integrated piece). In some aspects, the one or more conduits comprises a set of conduits connecting an outlet of the first reaction chamber to an inlet of the second reaction chamber. In some aspects, the conduit is capable of bi-directional flow. In some aspects, the one or more conduits comprises a first set of conduits connecting an outlet of the first reaction chamber to an inlet of the second reaction chamber, and a second set of conduits connecting an outlet of the second reaction chamber to an inlet of the first reaction chamber.
  • the flowing of the elongated oligonucleotide comprising the 3' blocking moiety to the second reaction chamber and/or the flowing of the elongated oligonucleotide without the 3' blocking moiety to the first reaction chamber is controlled by one or more pumps.
  • a pump is a solvent delivery system.
  • a pump can deliver a single solvent or multiple solvents.
  • the pump comprises a mixer that combines multiple solvents into a final solvent that can be delivered to the system. Pumps may be configured to deliver the solvents at a determined flow rate.
  • the one or more pumps deliver solvent at a constant flow rate.
  • the one or more pumps are configured to control a flow rate of the liquid phase.
  • the flow is controlled by a synthesizer.
  • the flowing of the elongated oligonucleotide comprising the 3' blocking moiety to the second reaction chamber and/or the flowing of the elongated oligonucleotide without the 3' blocking moiety to the first reaction chamber is controlled by one or more valves.
  • Valves are used for multiple purposes in a liquid delivery system.
  • Non-limiting examples of valves that can be used in the method include injection or inlet valves that are used to introduce liquid sample into the system, flow path switching valves that may be used to change the flow path of the liquid, column valves for attachment of one or more columns to the system, and outlet valves for waste or sample collection.
  • the one or more valves are multiposition rotary valves.
  • the valves are single position valves.
  • the method further comprises the use of one or more valves that selectively controls a flow pathway of the solution in the system.
  • the flowing of the elongated oligonucleotide comprising the 3' blocking moiety into the second reaction chamber by flowing the elongated oligonucleotide comprising the 3' blocking moiety through an outlet of the first reaction chamber.
  • the flowing of the elongated oligonucleotide comprising the 3' blocking moiety into the second reaction chamber comprising flowing the elongated oligonucleotide comprising the 3' blocking moiety through an inlet of the second reaction chamber.
  • the flowing of the elongated oligonucleotide without the 3' blocking moiety into the first reaction chamber comprises flowing the elongated oligonucleotide comprising the 3' blocking moiety through an outlet of the second reaction chamber.
  • the flowing of the elongated oligonucleotide without the 3' blocking moiety into the first reaction chamber comprises flowing the elongated oligonucleotide comprising the 3' blocking moiety through an inlet of the first reaction chamber.
  • the flowing the elongated oligonucleotide without the 3' blocking moiety from the second reaction chamber into the first reaction chamber comprises: flowing the elongated oligonucleotide without the 3' blocking moiety from the second reaction chamber into one or more reservoirs, and flowing the elongated oligonucleotide without the 3' blocking moiety from the one or more reservoirs into the first reaction chamber.
  • oligonucleotide comprising, in a plurality of reaction chambers : in at least a first reaction chamber, elongating an oligonucleotide in solution by attaching a first nucleotide comprising a 3' blocking moiety to the oligonucleotide using a first transferase to make an elongated oligonucleotide comprising the 3' blocking moiety; flowing the elongated oligonucleotide comprising the 3' blocking moiety in the solution from the at least the first reaction chamber into at least a second reaction chamber, wherein the first transferase is retained in the at least the first reaction chamber; in the at least the second reaction chamber, removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the 3' blocking moiety, the elongated
  • first transferase and the second transferase are different types of transferases.
  • the nucleotide comprising the 3' blocking moiety is a preselected type of nucleotide.
  • the first and second nucleotide are different pre-selected types of nucleotides.
  • first nucleotide and the second nucleotide are different types of nucleotides.
  • the 3' blocking moiety is removed from the elongated oligonucleotide comprising the 3' blocking moiety using a hydrolase, such as a phosphatase.
  • a hydrolase such as a phosphatase.
  • the hydrolase is an alkaline phosphatase.
  • the method further comprises separating the elongated oligonucleotide without the 3' blocking moiety from the hydrolase. Separating (e.g., substantially separating) can be done by immobilizing the hydrolase on a solid support, or using a filter or membrane to entrap or prevent passage of the hydrolase while the oligonucleotide flows through the pores.
  • the filter or membrane has a MWCO of a pre-determined value.
  • the method further comprises repeating the method for one or more cycles to further elongate the elongated oligonucleotide without the 3' blocking moiety.
  • the one or more cycles can be repeated at least once, at least twice, at least three, at least four, at least five, at least six times.
  • the method further comprises using a plurality of predetermined nucleotides are used to elongate the oligonucleotide, thereby generating an elongated oligonucleotide having a predetermined sequence, wherein each cycle after a first cycle attaches a single nucleotide to a previously elongated oligonucleotide without the 3' blocking moiety.
  • An oligonucleotide of a predetermined sequence may be generated by single addition of each pre-determined nucleotide to achieve said sequence.
  • the method further comprises using at least two different types of transferases in in separate cycles. Some transferases are better optimized to conjugate or ligate certain modified nucleotides to the oligonucleotide, due to reasons such as steric considerations that influence binding and/or efficient ligation.
  • the method comprises elongating the oligonucleotide in a first reaction chamber.
  • the method comprises removing the 3' blocking moiety in a second reaction chamber.
  • the hydrolase is retained in the at least the second reaction chamber when the elongated oligonucleotide without the 3' blocking moiety flows from the second reaction chamber into the at least the third reaction chamber.
  • the first reaction chamber, the second reaction chamber, and the third reaction chamber are connected through a plurality of conduits, wherein: the elongated oligonucleotide comprising the 3' blocking moiety flows from the at least the first reaction chamber into at least a second reaction chamber through a first portion of the plurality of conduits; and the elongated oligonucleotide without the 3' blocking moiety flows from the at least the second reaction chamber into at least a third reaction chamber through a second portion of the plurality of conduits.
  • a conduit such as a conduit that fluidly connects two reaction chambers, may be used to operably link two reaction chambers.
  • the one or more conduits operably links two or more reaction chambers.
  • conduits include chromatography tubing of any material, including glass, plastic, rubber, and silicone.
  • the conduit may be rigid, semi-rigid, or flexible.
  • a conduit may be assembled by connecting two or more conduits. Conduits may be connected using connectors, valves, or fittings.
  • a conduit may be monolithic (e.g., a single, integrated piece).
  • the one or more conduits comprises a set of conduits connecting an outlet of the first reaction chamber to an inlet of the second reaction chamber.
  • the conduit is capable of bi-directional flow.
  • the one or more conduits comprises a first set of conduits connecting an outlet of the first reaction chamber to an inlet of the second reaction chamber, and a second set of conduits connecting an outlet of the second reaction chamber to an inlet of the first reaction chamber.
  • the flowing of the elongated oligonucleotide comprising the 3' blocking moiety into the at least the second reaction chamber and/or the flowing of the elongated oligonucleotide without the 3' blocking moiety into the at least the third reaction chamber is controlled by one or more pumps.
  • a pump is a solvent delivery system.
  • a pump can deliver a single solvent or multiple solvents.
  • the pump comprises a mixer that combines multiple solvents into a final solvent that can be delivered to the system. Pumps may be configured to deliver the solvents at a determined flow rate.
  • the one or more pumps deliver solvent at a constant flow rate.
  • the one or more pumps are configured to control a flow rate of the liquid phase.
  • a synthesizer is a computer interface coupled with a flow system to automate synthesis, such as synthesis of an oligonucleotide. In some aspects, the flow is controlled by a synthesizer.
  • the flowing of the elongated oligonucleotide comprising the 3' blocking moiety into the at least the second reaction chamber and/or the flowing of the elongated oligonucleotide without the 3' blocking moiety into the at least the third reaction chamber is controlled by one or more valves.
  • Valves are used for multiple purposes in a liquid delivery system.
  • Non-limiting examples of valves that can be used in the method include injection or inlet valves that are used to introduce liquid sample into the system, flow path switching valves that may be used to change the flow path of the liquid, column valves for attachment of one or more columns to the system, and outlet valves for waste or sample collection.
  • the one or more valves are multiposition rotary valves.
  • the valves are single position valves.
  • the method further comprises the use of one or more valves that selectively controls a flow pathway of the solution in the system.
  • the nucleotide comprising a 3' blocking moiety is a nucleotide triphosphate comprising a 3' blocking moiety or an analog thereof comprising a 5' phosphate analog.
  • the 5' phosphate analog is a 5'-(a-P-thio)phosphate moiety .
  • the hydrolase removes the 3' blocking moiety from unreacted nucleotides in the solution. In some aspects, the hydrolase removes one or more 5' phosphate moieties and/or 5' phosphate analogs from unreacted nucleotides in the solution. In some aspects, the hydrolase removes three 5' phosphate moieties and/or 5' phosphate analogs from unreacted nucleotides in the solution.
  • the hydrolase is immobilized on a solid support .
  • the hydrolase is immobilized using a covalent, electrostatic, or ionic bond.
  • the transferase is a polymerase from the DNA polymerase X family. In some aspects, the transferase is a template independent transferase. In some aspects, the transferase is a terminal deoxynucleotidyl transferase (TdT). In some aspects, the transferase is immobilized on a solid support. In some aspects, the transferase is immobilized using a covalent, electrostatic, or ionic bond.
  • the method comprises comprising degrading the inorganic pyrophosphate using a pyrophosphatase.
  • the pyrophosphatase is an inorganic pyrophosphatase.
  • the pyrophosphatase is immobilized on a solid support.
  • the pyrophosphatase is immobilized using a covalent, electrostatic, or ionic bond.
  • the elongating and the degrading occur within the same reaction chamber.
  • the transferase and the inorganic pyrophosphatase are fused together.
  • the transferase and the pyrophosphatase are immobilized on the same solid support.
  • the transferase and the pyrophosphatase are immobilized on different solid supports.
  • the transferase is immobilized on a solid support and the pyrophosphatase is in the solution.
  • the pyrophosphatase is retained in the reaction chamber comprising the transferase, wherein the reaction chamber comprises a filter that prevents passage of the pyrophosphatase and allows passage of the oligonucleotide.
  • the elongating and the degrading occur within different reaction chambers.
  • the methods comprise flowing the elongated oligonucleotide comprising the 3' blocking moiety and the inorganic pyrophosphate byproduct, in the solution, from a reaction chamber comprising the transferase into a reaction chamber comprising the pyrophosphatase, followed by flowing the elongated oligonucleotide comprising the 3' blocking moiety from the reaction chamber comprising the pyrophosphatase into a reaction chamber in which the 3' blocking moiety is removed from the elongated oligonucleotide comprising the 3' blocking moiety.
  • the first reaction chamber or the second reaction chamber is a column.
  • the first reaction chamber comprises a fixed bed comprising the transferase immobilized on a solid support.
  • the first reaction chamber comprises a fluidized bed comprising the transferase immobilized on a solid support.
  • the first reaction chamber comprises a filter that prevents passage of the transferase and allows passage of the oligonucleotide.
  • the second reaction chamber comprises a fixed bed comprising the hydrolase immobilized on a solid support.
  • the second reaction chamber comprises a fluidized bed comprising the hydrolase immobilized on a solid support.
  • the second reaction chamber comprises a filter that prevents passage of hydrolase and allows passage of the oligonucleotide.
  • the first reaction chamber or the second reaction chamber is a batch reaction chamber.
  • the batch reaction chamber comprises an impeller.
  • the first reaction chamber comprises a rotating bed reactor comprising the transferase immobilized on a solid support.
  • the second reaction chamber comprises a rotating bed reactor comprising hydrolase immobilized on a solid support.
  • the methods further comprise separating unreacted nucleotides or reaction byproducts in the solution from the oligonucleotide.
  • the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using liquid chromatography.
  • the liquid chromatography comprises size exclusion chromatography.
  • the liquid chromatography comprises reverse phase chromatography.
  • the liquid chromatography comprises ion exchange chromatography.
  • the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using dialysis.
  • the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide using tangential flow filtration.
  • the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide after elongating the oligonucleotide by four or more nucleotides.
  • the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide only after elongating the oligonucleotide by four or more nucleotides.
  • the 3' blocking moiety is a phosphate moiety.
  • the nucleotide comprising the 3' blocking moiety is a ribonucleotide.
  • the nucleotide comprising the 3' blocking moiety is a deoxyribonucleotide.
  • the nucleotide comprising the 3' blocking moiety comprises a 2' modification.
  • the 2' modification is 2'-F or 2'-OMe.
  • the nucleotide comprising the 3' blocking moiety comprises a nucleoside 5'- (a-P-thio)phosphate.
  • the oligonucleotide comprises a 5' modification.
  • oligonucleotide comprising: elongating an oligonucleotide in solution by attaching a nucleotide comprising a 3' blocking moiety to the oligonucleotide using a transferase in the solution to make an elongated oligonucleotide comprising the 3' blocking moiety; separating the elongated oligonucleotide comprising the 3' blocking moiety from the transferase; removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety to make an elongated oligonucleotide without the 3' blocking moiety, the elongated oligonucleotide comprising the 3' blocking moiety being in solution when the 3' blocking moiety is removed.
  • separating the elongated oligonucleotide comprising the 3' blocking moiety from the transferase comprises flowing the solution through a column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety.
  • the column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety is a liquid chromatography column.
  • the column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety is a size exclusion column, an affinity column, an ion exchange column, or a reverse phase column.
  • the column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety comprises a filter that prevents passage of the transferase and allows passage of the oligonucleotide comprising the 3' blocking moiety.
  • the method further comprises flowing the oligonucleotide and the transferase, in the solution, from a reagent reservoir to the column that retains the transferase, and flowing the oligonucleotide comprising the 3' blocking moiety, in the solution, from the column that retains the transferase to the reagent reservoir.
  • removing the 3' blocking moiety from the elongated oligonucleotide comprising the 3' blocking moiety comprises adding a hydrolase to the solution after separating the elongated oligonucleotide comprising the 3' blocking moiety from the transferase.
  • the method further comprises separating the hydrolase from the elongated oligonucleotide without the 3' blocking moiety.
  • separating the hydrolase from the elongated oligonucleotide without the 3' blocking moiety from the hydrolase comprises flowing the solution through a column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety.
  • the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety is a liquid chromatography column. In some aspects, the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety is a size exclusion column, an affinity column, an ion exchange column, or a reverse phase column. In some aspects, the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety comprises a filter that prevents passage of the hydrolase and allows passage of the elongated oligonucleotide without the 3' blocking moiety.
  • the method comprises flowing the elongated oligonucleotide without the 3' blocking moiety and the hydrolase, in the solution, from a reagent reservoir to the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety, and flowing the elongated oligonucleotide without the 3' blocking moiety, in the solution, from the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety to the reagent reservoir.
  • the hydrolase is a phosphatase. In some aspects, the hydrolase is an alkaline phosphatase. In some aspects, the method comprises replacing or cleaning the column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety or the column that separates the hydrolase from the elongated oligonucleotide without the 3' blocking moiety. In some aspects, the cleaning comprises flowing a buffer through the column to elute the transferase or the hydrolase.
  • the method comprises separating unreacted nucleotides or reaction byproducts in the solution from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety.
  • separating unreacted nucleotides or reaction byproducts in the solution from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety comprises flowing the solution through a column that separates the unreacted nucleotides or reaction byproducts from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety.
  • the column that separates the unreacted nucleotides or reaction byproducts from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety is a liquid chromatography column. In some aspects, the column that separates the unreacted nucleotides or reaction byproducts from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety is a size exclusion column.
  • the column that separates the unreacted nucleotides or reaction byproducts from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety is a reverse phase column. In some aspects, the column that separates the unreacted nucleotides or reaction byproducts from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety is an ion exchange column.
  • the unreacted nucleotides or reaction byproducts are separated from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety using dialysis.
  • the unreacted nucleotides or reaction byproducts are separated from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety using tangential flow filtration.
  • the unreacted nucleotides or reaction byproducts are separated from the elongated oligonucleotide comprising the 3' blocking moiety or the elongated oligonucleotide without the 3' blocking moiety after elongating the oligonucleotide by four or more nucleotides.
  • the unreacted nucleotides or reaction byproducts are separated from the oligonucleotide only after elongating the oligonucleotide by four or more nucleotides.
  • the nucleotide comprising the 3' blocking moiety is a preselected type of nucleotide.
  • the nucleotide comprising a 3' blocking moiety is a nucleotide triphosphate comprising a 3' blocking moiety or an analog thereof comprising a 5' phosphate analog.
  • the 5' phosphate analog is a 5'-(a-P-thio)phosphate moiety.
  • the hydrolase removes the 3' blocking moiety from unreacted nucleotides in the solution.
  • the hydrolase removes one or more 5' phosphate moieties and/or 5' phosphate analogs from unreacted nucleotides in the solution.
  • the hydrolase removes three 5' phosphate moieties and/or 5' phosphate analogs from unreacted nucleotides in the solution.
  • the transferase is a polymerase from the DNA polymerase X family.
  • the transferase is a template independent transferase.
  • the transferase is a terminal deoxynucleotidyl transferase (TdT).
  • the elongating produces an inorganic pyrophosphate byproduct.
  • the method further comprises degrading the inorganic pyrophosphate using a pyrophosphatase.
  • the pyrophosphatase is an inorganic pyrophosphatase.
  • the transferase and the inorganic pyrophosphatase are fused together.
  • the column that separates the transferase from the elongated oligonucleotide comprising the 3' blocking moiety further separates the pyrophosphatase from the elongated oligonucleotide comprising the 3' blocking moiety.
  • the 3' blocking moiety is a phosphate moiety.
  • the nucleotide comprising the 3' blocking moiety is a ribonucleotide.
  • the nucleotide comprising the 3' blocking moiety is a deoxyribonucleotide.
  • the nucleotide comprising the 3' blocking moiety comprises a 2' modification.
  • the 2' modification is 2'-F or 2'-OMe.
  • the nucleotide comprising the 3' blocking moiety comprises a nucleoside 5'- (a-P-thio)phosphate.
  • the oligonucleotide comprises a 5' modification.
  • the oligonucleotide is elongated under an inert atmosphere. In some aspects, the oligonucleotide is deblocked under an inert atmosphere. In some aspects, the inert atmosphere is maintained by an inert gas.
  • elongating the oligonucleotide is performed between 35 °C and 45 °C. In some aspects, removing the 3' blocking moiety is performed between 45 °C and 55 °C.
  • the elongated oligonucleotide without the 3' blocking moiety is buffer exchanged and/or concentrated using a desalting chamber.
  • the desalting chamber is maintained between 20 °C and 30 °C.
  • the cultures were incubated for approximately 195 min at 30 °C, 250 rpm, to an ODeoo of about 0.6, and then induced with the addition of IPTG at a final concentration of 1 mM.
  • the induced cultures were incubated for 20 h at 30 °C, 250 rpm. Following this incubation period, the cultures were centrifuged at 4000 rpm x 10 min. The culture supernatant was discarded, and the pellets were resuspended in 35 ml of 20 mM triethanolamine, pH 7.5.
  • This cell suspension was chilled in an ice bath and lysed using a Microfluidizer cell disruptor (Microfluidics M-l 10L). The crude lysate was pelleted by centrifugation (11,000 rpm for 60 min at 4 °C), and the supernatant was then filtered through a 0.2 pm PES membrane to further clarify the lysate.
  • the clarified lysates were then purified using an AKTA Start purification system and a 5 mL HisTrap FF column (GE Healthcare) using the AC Step HiF setting (the run parameters are provided below in Table 1).
  • the SF wash buffer comprised 50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, 0.02% v/v Triton X-100 reagent.
  • Elution fractions containing protein were identified by UV absorption (A280) and pooled, then dialyzed overnight in dialysis buffer (20 mM Tris-HCl, pH 7.4, 100 mM KC1, 0.1 mM EDTA, and 50% glycerol) in a 3.5K Slide-A-LyzerTM dialysis cassette (Thermo Fisher) for buffer exchange. iPP concentrations from the preparations were measured by absorption at 280 nm.
  • Example 2 Sample preparation for analysis by capillary electrophoresis (CE) analysis of oligonucleotide reaction products
  • capillary electrophoresis was performed using an ABI 3500x1 Genetic Analyzer (ThermoFisher). Reactions (1 uL) were quenched by the addition of 99 L of 1 mM aqueous EDTA. Quenched reactions were diluted in water to 1.25 nM oligonucleotide, and 2 pL of this solution was transferred to a new 96-well MicroAmp Optical PCR plate or 384-well Micro Amp Optical PCR plate containing 18 pL Hi-DiTM Formamide (ThermoFisher) containing an appropriate size standard (LEZ or Alexa633).
  • the ABI3500xl was configured with POP6 polymer, 50 cm capillaries and a 55 °C oven temperature. Pre-run settings were 18KV for 50 sec. Injection was 10KV for 2 sec, and the run settings were 19 KV for 620 sec. FAM-labeled oligo substrates and products were identified by their sizes relative to the sizing ladder.
  • Example 3 High performance liquid chromatography (HPLC) analysis of nucleotides
  • the 5 to 20 pL crude reaction product is diluted to a final total nucleotide concentration of 100 pM nucleotide using 1 mM EDTA in RO water as diluent. 100 pL is transferred from the supernatant to a 96 well round bottom plate and then 10 pL is injected onto an Ultimate 3000 HPLC system using a PAL autosampler according to the method outlined in Table 3.1.
  • a 2 to 10 pL aliquot is removed from the reaction and diluted to a concentration of 25 pM oligonucleotide using 1 mM EDTA (pH 8) in RO water as diluent. 10 pL from the diluted samples is then injected onto an Ultimate 3000 HPLC system using a PAL autosampler according to either Method A outlined in Table 4.1, or Method B outlined in Table 4.2.
  • Residual alkaline phosphatase is measured by reaction with p-nitrophenyl phosphate.
  • a 100 pL diluted sample containing 2.0 pM enzyme is prepared, using the same buffer as the analyte as diluent.
  • 20 pL from the 2.0 pM stock is serially diluted by 2x across one row of a biorad 96-well PCR plate using analyte buffer as diluent, with a final volume of 10 pL in each well (final concentrations ranging from 2.0 to 0.001 pM enzyme).
  • 10 pL is removed from each sample to be analyzed and transferred to one well of the biorad 96-well PCR plate.
  • a reaction buffer stock is then prepared containing: p-nitrophenylphosphate (2.0 mM), CoCf (1.0 mM), TEoAHCl (50 mM, pH 7.8).
  • 10 pL is transferred from the reaction stock to the wells containing analyte and serially diluted AP on the biorad 96-well PCR plate.
  • the plate is heat sealed with an aluminum seal, mixed, and briefly centrifuged to collect the liquid in the bottom of each well.
  • the plate is then heated at 50 °C for 10 minutes on a thermal cycler. Afterward, the reaction is quenched by diluting each well with 100 pL of 1.0 M NaOH.
  • the samples are transferred to a clear flat-bottom 96-well plate for analysis by UV-Vis absorbance at 405 nm on a SpectraMax M2 plate reader.
  • a linear least-squares fitting to the linear portion of the AP dose response curve from 0.0005 pM to 0.05 pM is used to measure the amount of AP in the analyte wells.
  • a 10 mM C0CI2 solution was prepared in the same buffer as the analyte to be assayed and serially diluted across 12 wells of a clear flat bottom plate so that each well had 50 pL and the last well was blank. A 100 pL aliquot was removed from each sample to be measured and diluted 2x across three wells in the same clear bottom plate so that each sample well contained 50 pL.
  • a working reagent stock was prepared using solutions A, B, and C from a Micro BCATM protein assay kit purchased from Thermo Scientific according to the directions provided.
  • TnT or AP was first produced in shake flask and purified as described in Example 1.
  • the storage buffer from an aliquot of the desired protein stock was then exchanged for TEAHC1 (500 mM, pH 7.8) by diluting lOx with TEAHC1 followed by concentration through a Sartirous VivaSpin 6 (30,000 MWCO) spin filter at 4000 rpm and 4 °C.
  • the resulting small volume is then diluted another 2.5 x with TEAHC1 (500 mM, pH 7.8) to give the original volume.
  • affinity resin was weighed out, either wet or dry, into a 96 well plate with a volume of 2 mL per well or into a 2 mL Eppendorf tube.
  • 0.5 to 1.0 g adsorbent or ion-exchange resin was weighed out into a 15 mL conical tube.
  • the volume of protein stock containing the desired wt. % of protein vs resin mass was transferred to the 96 well plate or tube containing resin.
  • the resin was collected by vacuum filtration and then washed three times with 10 mL of TEAHC1 (50 mM, pH 7.8), allowing the resin to mix well on a rotator for 10 minutes at room temperature during each wash.
  • TEAHC1 50 mM, pH 7.8
  • TnT enzyme variant (SEQ ID NO: 2) was produced and purified according to Example 1.
  • iPP enzyme variant (SEQ ID NO: 8) was produced and purified according to Example 1.
  • reaction stocks comprising four oligo concentrations matrixed with two CoCE concentrations were prepared, each containing: 5’-AAAAmC*mA*mG (800, 400, 200, or 100
  • reaction stocks comprising four oligonucleotide concentrations matrixed with three CoCh concentrations were prepared, each containing: 5’-AAAAfGmUfG (3200, 1600, 800, or 400 pM), mUTP-3’P (2.0 equivalents vs oligo), C0CI2 (2.0, 1.0, or 0.5 mM), TEoAHCl (50 mM), pH 7.8.
  • reaction buffer vs resin wet weight 20 pL/mg of reaction buffer vs resin wet weight. The resins were incubated with reaction buffer for 24 h at 50 °C and 500 rpm agitation. A 10 pL aliquot was then removed from each reaction and diluted to 25 pM oligonucleotide in 1 mM EDTA for analysis by HPLC according to Example 4, Method A. A second 10 pL aliquot was removed and diluted to 100 pM nucleotide using 1 mM EDTA and analyzed by HPLC according to Example 4.
  • AU Cfotai representative of the percent of total remaining nucleotide that is constituted of unhydrolyzed mUTP- 3’P and does not take into account mUTP-3’P consumed in the reaction with TnT and oligonucleotide.
  • reaction stocks comprising four oligonucleotide concentrations matrixed with three CoCh concentrations were prepared, each containing: 5’-AAAAfGmUfG (3200, 1600, 800, or 400 pM), mUTP-3’P (2.0 equivalents vs oligo), C0CI2 (2.0, 1.0, or 0.5 mM), TEoAHCl (50 mM), pH 7.8.
  • reaction buffer vs resin wet weight 20 pL/mg of reaction buffer vs resin wet weight. The resins were incubated with reaction buffer for 24 h at 50 °C and 500 rpm agitation. A 10 pL aliquot was then removed from each reaction and diluted to 25 pM oligonucleotide in 1 mM EDTA for analysis by HPLC according to Example 4, Method A. A second 10 pL aliquot was removed and diluted to 100 pM nucleotide using 1 mM EDTA and analyzed by HPLC according to Example 3.
  • the conversion of 5’-AAAAfGmUfG to 5’-AAAAfGmUfGmU-3’P along with the percent unhydrolyzed mUTP-3’P in the reaction stock after 24h is reported in Table 9.3.
  • the percent unhydrolyzed mUTP-3’P is calculated as the AUCmU p ⁇ 3,p taken from the HPLC chromatogram, representative of the percent of total remaining nucleotide that is constituted of unhydrolyzed mUTP- 3’P and does not take into account mUTP-3’P consumed in the reaction with TnT and oligonucleotide.
  • Example 10 Co-Immobilized TnT SEQ ID NO: 2/iPP SEQ ID NO: 8 and TnT SEQ ID NO: 4/iPP SEQ ID NO: 8 Enzymatic Extension of 5’-AAAAfGmUfGmU
  • TnT enzyme variant (SEQ ID NO: 2) was produced and purified according to Example 1.
  • TnT enzyme variant (SEQ ID NO: 4) was produced and purified according to Example 1.
  • iPP enzyme variant (SEQ ID NO: 8) was produced and purified according to Example 1.
  • TnT (SEQ ID NO: 4) and iPP were co-immobilized 2.5 wt% and 0.2 wt% vs wet weight of resin, respectively, according to Example 8.
  • TnT (SEQ ID NO: 4) and iPP were co-immobilized 2.5 wt% and 0.02 wt% vs wet weight of resin, respectively, according to Example 8. After washing, the resins were transferred to 8 wells of a 1.0 mL deep 96-well plate.
  • Alkaline phosphatase SEQ ID NO: 10 was produced and purified, as described in Example 1 and as described in patent application No. PCT/US2023/076667, filed October 12, 2023, which is incorporated by reference herein in its entirety.
  • the AP was diluted lOx into 20 mM MOPS (pH 7.0) then concentrated through a 10 kDa spin filter. The concentrated stock was diluted further with 20 mM MOPS (pH 7.0) to produce a 0.1 g/L and a 1.0 g/L master stock.
  • Each sample was washed with one of three wash methods: (1) Three 150 pL washes of 20 mM MOPS (pH 7.0); (2) 300 pL of 1 M ethanolamine in 20 mM MOPS (pH 7.0) for 4 h at room temperature followed by three 150 pL washes of 20 mM MOPS (pH 7.0); or (3) 300 pL of 50 mM sodium phosphate (NaPi) buffer for 24 h at 4 °C followed by three 150 pL washes of 20 mM MOPS (pH 7.0).
  • a reaction stock was prepared consisting of: 5’-(FAM)Ti5mAmG*mGmA-3’P (0.225 pM), 5’-Ti 5 mAmG*mGmA-3’P (199.8 pM), CoCh (0.25 mM), TEoAHCl (20 mM), pH 7.8.
  • a volume of reaction stock was added such that the final on-resin AP was 0.4 or 4.0 mg/mL for resins loaded at a targeted 0.25 or 2.5 weight percent vs wet weight of resin, respectively.
  • the samples were then heated at 50 °C for 15 minutes.
  • Alkaline phosphatase (AP) enzyme SEQ ID NO: 12 was produced in shake flask and purified as described in Example 1.
  • Alkaline phosphatase (AP) enzyme variant (SEQ ID NO: 12) was produced in shake flask and purified as described in Example 1.
  • a reaction solution comprised of 800 pM 5’-AAAAmC*mA*mGmA-3’P, residual mATP- 3’P, 1.0 mM CoCL, and 220 pM iPP in TEAHC1 (50 mM, pH 7.8) was prepared.
  • a 300-500 pL portion of this reaction solution was added to each of the 2 mL Eppendorf tubes containing epoxide functionalized resins loaded with 2.0 wt. % AP treated with a post-immobilization quench reagent.
  • the reaction was then shaken at 45 °C, 600 rpm for 1 h. After the reaction was complete, a 5 uL aliquot of the reaction was diluted to 160 pL in 1 mM EDTA and analyzed via the HPLC method as described in Example 4, Method B.
  • TnT enzyme variant (SEQ ID NO: 4) was produced and purified according to Example 1.
  • iPP enzyme variant (SEQ ID NO: 8) was produced and purified according to Example 1.
  • TnT and iPP were immobilized on Chiral Vision IB -HIS -2 resin according to Example 8.
  • a reaction stock (4 mL) was prepared containing: 5’-AAAAmC*mA*mG (1 mM), mATP- 3’P (1.5 mM) CoCl 2 (1.0 mM), TEAHC1 (50 mM), pH 7.8.
  • This reaction master mix was added to the plastic tube containing the resin loaded spin reactor. The reaction was then incubated at 50 °C for 4 h with magnetic stirring at 400 rpm.
  • ChemSpin MagRBR spin reactors loaded with 250 mg IB-COV-7 resin, charged with 2 and 1 wt. % AP were loaded into two of the plastic tubes containing the reaction mixture resulting from the extension reaction described above. AP was added to the remaining plastic tube such that the same amount of enzyme was added in the tube as was immobilized on the 2 wt% sample. The reaction was then incubated at 50 °C for 2 h at 400 rpm.
  • TnT variant (SEQ ID NO: 4) and IPP variant (SEQ ID NO: 8) were co-immobilized on ChiralVision IB-HIS-2 resin, pre-loaded with CoCL. at 2.5 and 0.02 wt. % vs wet weight of resin, respectively, according to Example 8. Additional preparations of TnT and iPP co-immobilized on ChiralVision IB-HIS-2 resin at 2.5 and 0.2 wt. % were prepared according to Example 8 as needed.
  • a BioRad Econo Alpha column (manufacturer part # 12009463) with internal dimensions of 6.6 mm x 50 mm (650 pL internal volume), fitted with an adjustable flow adapter containing 5 um pore size frits, was packed with 450 mg of TnT and iPP co-immobilized on ChiralVision IB-HIS-2 resin at 2.5 and 0.02 wt. %, respectively; a small portion of TEAHC1 (50 mM, pH 7.8) was used to aid in the transfer of resin to the column.
  • the flow adapter was used to compress the resin to give a bed volume of approximately 500 pL.
  • the column was fitted with a resistive heating jacket to maintain a bed temperature of 40-60 °C.
  • reaction solution was flushed from the system via peristaltic pump and the column washed with TEAHC1 (50 mM, pH 7.8) to collect retained RNA oligomer. Aliquots of the wash buffer were analyzed by HPLC for the presence of RNA oligomer as described in Example 4.
  • Example 16 Procedure for Enzymatic 3-Dephosphorylation of an RNA Oligomer Using Immobilized Alkaline Phosphatase in a Single-Pass Flow Reactor
  • Alkaline phosphatase was immobilized on ChiralVision IB-COV-7 resin at 2.0 wt. % vs wet weight of resin according to Example 11. Additional preparations of alkaline phosphatase immobilized on ChiralVision IB-COV-7 resin at 2.0 wt. % were prepared according to Example 11 as needed.
  • a BioRad Econo Alpha column (manufacturer part # 12009463) with internal dimensions of 6.6 mm x 50 mm (650 pL internal volume), fitted with an adjustable flow adapter containing 5 um pore size frits, was packed with 450 mg of alkaline phosphatase immobilized on ChiralVision IB- COV-7 resin at 2.0 wt. %: a small portion of TEAHC1 (50 mM, pH 7.8) was used to aid in the transfer of resin to the column.
  • the flow adapter was used to compress the resin to give a bed volume of approximately 500 pL.
  • the column was fitted with a resistive heating jacket to maintain a bed temperature of 40-60 °C.
  • a 15 mL Falcon tube containing the crude extension reaction mix comprised of 50 mM TEAHC1 buffer at pH 7.8, 100-1600 pM RNA oligomer, 0.5-2.0 mM CoCl 2 , and 200-3200 pM NTP- 3’P, was pumped via peristaltic pump at a flow rate of 0.04-0.50 mL/min through the temperature- controlled column containing alkaline phosphatase immobilized at 2.0 wt. % on ChiralVision IB- COV-7. After passing through the column containing immobilized alkaline phosphatase, the solution was collected in a separate collection vessel. The endpoint of the reaction was determined by analyzing an aliquot removed from the reservoir via HPLC, as described in Example 4. Residual NTP-3’P dephosphorylation was analyzed by the HPLC method as described in Example 3.
  • Example 17 Leaching of Alkaline Phosphatase Immobilized on Epoxy Resin in a Single Pass Flow Reactor
  • Alkaline phosphatases SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 were immobilized onto IB-COV-7 according to Example 11 and packed into a BioRad Econo Alpha column according to Example 16.
  • Example 18 Activity of Alkaline Phosphatase Immobilized on Epoxy Resin in a Single Pass Flow Reactor
  • Activity relative to SEQ ID NO: 10 was calculated as the percent mATP-3’P of the variant compared with the percent mATP-3’P observed by the reaction with SEQ ID NO: 10. The results are shown in Table 18.1.
  • TnT enzyme variant (SEQ ID NO: 4) was produced in shake flask and purified as described in Example 1.
  • Inorganic pyrophosphatase (iPP) variant (SEQ ID NO: 8) as produced and purified, as described in Example 1.
  • Alkaline phosphatase (AP) variant (SEQ ID NO: 10) was produced and purified, as described in Example 1.
  • TnT and iPP were co-immobilized on ChiralVision IB-HIS-2 resin (3 g), pre-loaded with C0CI2, at 2.5 and 0.02 wt. % vs wet weight of resin, respectively, according to Example 8.
  • AP was immobilized on Chiral Vision IB-COV-7 resin (3 g) at 2.0 wt. % protein vs wet weight of resin, according to Example 11.
  • a BioRad Econo Alpha column was packed with TnT and iPP co-immobilized on ChiralVision IB-HIS-2 at 2.5 and 0.02 wt. %, respectively, according to the procedure in Example 15.
  • the column bed was maintained at 40 °C via restive heating jacket throughout the duration of the experiment.
  • the reaction solution was recirculated through the packed resin bed using a peristaltic pump with a flow rate of 0.11 mL/min for 19 h until the reaction had reached completion via the HPLC method described in Example 4, Method A.
  • a BioRad Econo Alpha column was packed with TnT and iPP co-immobilized on ChiralVision IB-HIS-2 at 2.5 and 0.02 wt. %, respectively, according to the procedure in Example 15.
  • the column bed was maintained at 40 °C via restive heating jacket throughout the duration of the experiment.
  • the reaction solution was recirculated through the packed resin bed using a peristaltic pump with a flow rate of 0.11 mL/min for 1.5 h until the reaction had reached completion as determined by the HPLC method described in Example 4, Method A.
  • a BioRad Econo Alpha column was packed with TnT and iPP co-immobilized on ChiralVision IB-HIS-2 at 2.5 and 0.02 wt. %, respectively, according to the procedure in Example 15.
  • the column bed was maintained at 40 °C via restive heating jacket throughout the duration of the experiment.
  • the reaction solution was recirculated through the packed resin bed using a peristaltic pump with a flow rate of 0.11 mL/min for 18.5 h until the reaction had reached completion as determined by the HPLC method described in Example 4 Method A.
  • a BioRad Econo Alpha column was packed with TnT and iPP co-immobilized on ChiralVision IB-HIS-2 at 2.5 and 0.02 wt. %, respectively, according to the procedure in Example 15.
  • the column bed was maintained at 40 °C via restive heating jacket throughout the duration of the experiment.
  • the reaction solution was recirculated through the packed resin bed using a peristaltic pump with a flow rate of 0.11 mL/min for 2.5 h until the reaction had reached completion as determined by the HPLC method described in Example 4, Method A.
  • a BioRad Econo Alpha column was packed with TnT and iPP co-immobilized on ChiralVision IB-HIS-2 at 2.5 and 0.02 wt. %, respectively, according to the procedure in Example 15.
  • the column bed was maintained at 40 °C via restive heating jacket throughout the duration of the experiment.
  • the reaction solution was recirculated through the packed resin bed using a peristaltic pump with a flow rate of 0.11 mL/min for 7 h until the reaction had reached completion as determined by the HPLC method described in Example 4, Method A
  • a BioRad Econo Alpha column was packed with TnT and iPP co-immobilized on ChiralVision IB-HIS-2 at 2.5 and 0.02 wt. %, respectively, according to the procedure in Example 15.
  • the column bed was maintained at 40 °C via restive heating jacket throughout the duration of the experiment.
  • the reaction solution was recirculated through the packed resin bed using a peristaltic pump with a flow rate of 0.11 mL/min for 5 h until the reaction had reached completion as determined by the LCMS method described in Example 5.
  • a BioRad Econo Alpha column was packed with AP immobilized on ChiralVision IB-COV-7 at 2.0 wt. % according to the procedure in Example 16.
  • the column bed was maintained at 50 °C via restive heating jacket throughout the duration of the experiment.
  • the reaction solution was pumped through the packed resin bed using a peristaltic pump with a flow rate of 0.06 mL/min until the entire reaction solution had been pumped through the resin bed and collected in a separate vessel.
  • the level of 3’P-dephosphorylation was measured via HPLC as described in Example 4, Method A.
  • the residual AP activity of the final reaction solution was measured as described in Example 6.
  • the residual CoCL concentration of the final reaction solution was measured according to the method described in Example 7 and adjusted to 1.0 mM final C0CI2 concentration prior to moving on to the next cycle.
  • a BioRad Econo Alpha column was packed with AP immobilized on ChiralVision IB-COV-7 at 2.0 wt. % according to the procedure in Example 16.
  • the column bed was maintained at 50 °C via restive heating jacket throughout the duration of the experiment.
  • the reaction solution was pumped through the packed resin bed using a peristaltic pump with a flow rate of 0.06 mL/min until the entire reaction solution had been pumped through the resin bed and collected in a separate vessel.
  • the level of 3’P-dephosphorylation was measured via HPLC as described in Example 4, Method A.
  • the residual AP activity of the final reaction solution was measured as described in Example 6.
  • the residual C0CI2 concentration of the final reaction solution was measured according to the method described in Example 7 and adjusted to 1.0 mM final C0CI2 concentration prior to moving on to the next cycle.
  • a BioRad Econo Alpha column was packed with AP immobilized on ChiralVision IB-COV-7 at 2.0 wt. % according to the procedure in Example 16.
  • the column bed was maintained at 50 °C via restive heating jacket throughout the duration of the experiment.
  • the reaction solution was pumped through the packed resin bed using a peristaltic pump with a flow rate of 0.06 mL/min until the entire reaction solution had been pumped through the resin bed and collected in a separate vessel.
  • the level of 3’P-dephosphorylation was measured via HPLC as described in Example 4, Method A. Dephosphorylation of residual mUTP-3’P was measured by the HPLC method as described in Example 3.
  • the residual AP activity of the final reaction solution was measured as described in Example 6.
  • the residual C0CI2 concentration of the final reaction solution was measured according to the method described in Example 7 and adjusted to 1.0 mM final C0CI2 concentration prior to moving on to the next cycle.
  • the residual AP activity of the final reaction solution was measured as described in Example 6.
  • the residual C0CI2 concentration of the final reaction solution was measured according to the method described in Example 7 and adjusted to 1.0 mM final C0CI2 concentration prior to moving on to the next cycle.
  • a BioRad Econo Alpha column was packed with AP immobilized on ChiralVision IB-COV-7 at 2.0 wt. % according to the procedure in Example 16.
  • the column bed was maintained at 50 °C via restive heating jacket throughout the duration of the experiment.
  • the reaction solution was pumped through the packed resin bed using a peristaltic pump with a flow rate of 0.06 mL/min until the entire reaction solution had been pumped through the resin bed and collected in a separate vessel.
  • the level of 3’P-dephosphorylation was measured via HPLC as described in Example 4, Method A. Dephosphorylation of residual fCTP-3’P was measured by the HPLC method as described in Example 3.
  • the residual AP activity of the final reaction solution was measured as described in Example 6.
  • the residual C0CI2 concentration of the final reaction solution was measured according to the method described in Example 7 and adjusted to 1.0 mM final C0CI2 concentration prior to moving on to the next cycle.
  • a BioRad Econo Alpha column was packed with AP immobilized on ChiralVision IB-COV-7 at 2.0 wt. % according to the procedure in Example 16.
  • the column bed was maintained at 50 °C via restive heating jacket throughout the duration of the experiment.
  • the reaction solution was pumped through the packed resin bed using a peristaltic pump with a flow rate of 0.06 mL/min until the entire reaction solution had been pumped through the resin bed and collected in a separate vessel.
  • the level of 3’P-dephosphorylation was measured via HPLC as described in Example 4, Method A.
  • the residual AP activity of the final reaction solution was measured as described in Example 6.
  • the residual C0CI2 concentration of the final reaction solution was measured according to the method described in Example 7 and adjusted to 1.0 mM final CoCf concentration prior to moving on to the next cycle.
  • TnT enzyme variant (SEQ ID NO: 4) was produced in shake flask and purified as described in Example 1.
  • Inorganic pyrophosphatase (iPP) variant (SEQ ID NO: 8) was produced and purified, as described in Example 1.
  • the jacket temperature was set to 4 °C and the resin was gently stirred at 8 rpm for 20 h until the change in A280 of the immobilization solution became constant.
  • the immobilization solution and resin were drained from the jacketed reaction vessel and the resin was collected on a coarse glass filter via vacuum filtration.
  • the resin was washed with four 250 mL volumes of TEAHC1 (50 mM, pH 7.8). The resin was allowed to dry on the filter while maintaining vacuum for approximately ten minutes, then transferred to a sealed plastic storage container and stored at 4 °C.
  • Alkaline phosphatase (AP) variant (SEQ ID NO: 10) was produced and purified, as described in Example 1.
  • the jacket temperature was set to 4 °C and the resin was gently stirred at 8 rpm for 20 h until the change in A280 of the immobilization solution became constant.
  • the immobilization solution and resin were drained from the jacketed reaction vessel and the resin collected on a coarse glass filter via vacuum filtration.
  • the resin was washed with five 200 mL volumes of TEAHC1 (50 mM, pH 7.8) containing 500 mM NaCl, followed by five additional 200 mL wash volumes of TEAHC1 (50 mM, pH 7.8).
  • the resin was allowed to dry on the filter while maintaining vacuum for approximately ten minutes, then transferred to a sealed plastic storage container and stored at 4 °C.
  • TnT and IPP were co-immobilized on ChiralVision IB-HIS-2 resin, pre-loaded with CoCU, at 2.5 and 0.02 wt. % vs wet weight of resin, respectively, according to Example 20. Additional preparations of TnT and iPP co-immobilized on ChiralVision IB-HIS-2 resin at 2.5 and 0.2 wt. % were prepared according to Example 20 as needed.
  • a Cytiva XK 16/40 jacketed column (manufacturer part # 28988938) with internal dimensions of 16 mm x 400 mm (80 mL internal volume) fitted with two Cytiva adjustable flow adapters (manufacturer part # 28989876) was packed with 20.3 g of TnT and iPP co-immobilized on ChiralVision IB-HIS-2 resin at 2.5 and 0.02 wt. %, respectively; a portion of TEAHC1 (50 mM, pH 7.8) was used to aid in the transfer of resin to the column.
  • the flow adapters were used to compress the resin to give a bed volume of approximately 29 mL.
  • the column bed temperature was maintained with a recirculating water heater at 40-60 °C throughout the course of the extension reaction.
  • reaction solution was flushed from the system via peristaltic pump and the column washed with TEAHC1 (50 mM, pH 7.8) to collect retained RNA oligomer. Aliquots of the wash buffer were analyzed by HPLC for the presence of RNA oligomer as described in Example 4, Method A.
  • Alkaline phosphatase was immobilized on ChiralVision IB-COV-7 resin at 2.0 wt. % vs wet weight of resin according to Example 21. Additional preparations of alkaline phosphatase immobilized on ChiralVision IB-COV-7 resin at 2.0 wt. % were prepared according to Example 21 as needed.
  • a Cytiva XK 16/40 jacketed column (manufacturer part # 28988938) with internal dimensions of 16 mm x 400 mm (80 mL internal volume) fitted with two Cytiva adjustable flow adapters (manufacturer part # 28989876) was packed with 20.3 g of alkaline phosphatase immobilized on ChiralVision IB-COV-7 resin at 2.0 wt. %; a portion of TEAHC1 (50 mM, pH 7.8) was used to aid in the transfer of resin to the column.
  • the flow adapter was used to compress the resin to give a bed volume of approximately 27 mL.
  • the column bed temperature was maintained with a recirculating water heater at 40-60 °C throughout the course of the 3’P-dephosphorylation reaction.
  • a 250-1000 mL flask containing the crude extension reaction mix comprised of 50 mM TEAHC1 buffer at pH 7.8, 100-3000 pM RNA oligomer, 0.5-2.0 mM CoCl 2 , and 200-6000 pM NTP- 3’P, was pumped via peristaltic pump at a flow rate of 0.04-5.00 mL/min first through an inline degasser, then through the temperature-controlled column containing alkaline phosphatase immobilized at 2.0 wt. % on ChiralVision IB-COV-7. After passing through the column containing immobilized alkaline phosphatase, the solution was collected in a separate collection vessel. The endpoint of the reaction was determined by analyzing an aliquot removed from the reservoir via HPLC, as described in Example 4, Method A. Residual NTP-3’P dephosphorylation was analyzed by the HPLC method as described in Example 3.
  • TnT enzyme variants SEQ ID NO: 4 and SEQ ID NO: 2 were produced in shake flask and purified as described in Example 1.
  • Inorganic pyrophosphatase (iPP) variant (SEQ ID NO: 8) was produced and purified, as described in Example 1.
  • Alkaline phosphatase (AP) variant (SEQ ID NO: 10) was produced and purified, as described in Example 1.
  • TnT variant (SEQ ID NO: 4) and iPP variant (SEQ ID NO: 8) were co-immobilized on 110 g of ChiralVision IB-HIS-2 resin pre-loaded with CoCL, at 2.5 and 0.02 wt. % vs wet weight of resin, respectively, according to Example 20.
  • TnT variant (SEQ ID NO: 2) and iPP variant (SEQ ID NO: 8) were co-immobilized on 20 g of ChiralVision IB-HIS-2 resin, pre-loaded with C0CI2, at 2.5 and 0.02 wt. % vs wet weight of resin, respectively, according to Example 8.
  • AP was immobilized on 110 g ChiralVision IB-COV-7 resin at 2.0 wt. % protein vs wet weight of resin, according to Example 21.
  • a Cytiva XK16-40 column was packed with 50 mL of Ni Sepharose 6 Fast Flow resin in series with two Bio-Rad EconoFit 5 mL columns packed with un-metalated Sepharose 6 Fast Flow resin to prevent Ni leaching. Protein was removed from the reaction mixture by passing through the columns at 5 mL/min. The column was washed with 40 mL of 50 mM TEoAHCl, pH 7.8, giving a final solution volume of 160 mL.
  • reaction solution in a 250 mL conical flask containing a 180 mL solution comprised of 2.25 mM 5’-AAAAfGmUfGmU and 1 mM C0CI2 in TEAHC1 (50 mM, pH 7.8) was added 4.5 mM of fCTP-3’P.
  • the reaction solution was maintained at 4 °C throughout the duration of the extension reaction.
  • a Cytiva XK 16/40 column was packed with 20.3 g of TnT variant (SEQ ID NO: 4) and iPP co-immobilized on ChiralVision IB-HIS-2 at 2.5 and 0.02 wt. %, respectively, according to the procedure in Example 22.
  • the column bed was maintained at 40 °C via heated water jacket throughout the duration of the experiment.
  • the reaction solution was recirculated through the packed resin bed using a peristaltic pump with a flow rate of 10.2 column volumes per hour until the reaction had reached completion as determined by the HPLC method described in Example 4, Method A.
  • a Cytiva XK 16/40 column was packed with 20.3 g of TnT variant (SEQ ID NO: 2) and iPP co-immobilized on ChiralVision IB-HIS-2 at 2.5 and 0.02 wt. %, respectively, according to the procedure in Example 22.
  • the column bed was maintained at 40 °C via heated water jacket throughout the duration of the experiment.
  • the reaction solution was recirculated through the packed resin bed using a peristaltic pump with a flow rate of 10.2 column volumes per hour for 24 h.
  • a Cytiva XK 16/40 column was packed with 20.3 g of TnT variant (SEQ ID NO: 4) and iPP co-immobilized on ChiralVision IB-HIS-2 at 2.5 and 0.02 wt. %, respectively, according to the procedure in Example 22.
  • the column bed was maintained at 40 °C via heated water jacket throughout the duration of the experiment.
  • the reaction solution was recirculated through the packed resin bed using a peristaltic pump with a flow rate of 10.2 column volumes per hour until the reaction had reached completion as determined by the HPLC method described in Example 4, Method A.
  • a Cytiva XK 16/40 column was packed with 20.3 g of TnT variant (SEQ ID NO: 4) and iPP co-immobilized on ChiralVision IB-HIS-2 at 2.5 and 0.02 wt. %, respectively, according to the procedure in Example 22.
  • the column bed was maintained at 40 °C via heated water jacket throughout the duration of the experiment.
  • the reaction solution was recirculated through the packed resin bed using a peristaltic pump with a flow rate of 10.2 column volumes per hour until the reaction had reached completion as determined by the HPLC method described in Example 4, Method A.
  • RNA oligomer [0500] The system was purged of remaining reaction solution and the column washed with approximately two column volumes of TEAHC1 (50 mM, pH 7.8) to remove any retained RNA oligomer from the system.
  • the system wash step was determined to be complete when aliquots of the wash solution contained no RNA oligomer via the HPLC method described in Example 4, Method A.
  • the wash solution containing RNA oligomer was added to the reaction reservoir giving a final volume of 597 mL, and enzymatic 3 ’-dephosphorylation with immobilized alkaline phosphatase as outlined below was carried out.
  • a Cytiva XK 16/40 column was packed with 20.3 g of TnT variant (SEQ ID NO: 4) and iPP co-immobilized on ChiralVision IB-HIS-2 at 2.5 and 0.02 wt. %, respectively, according to the procedure in Example 22.
  • the column bed was maintained at 40 °C via heated water jacket throughout the duration of the experiment.
  • the reaction solution was recirculated through the packed resin bed using a peristaltic pump with a flow rate of 10.2 column volumes per hour until the reaction had reached completion as determined by the HPLC method described in Example 4, Method A.
  • a Cytiva XK 16/40 column was packed with 20.3 g of TnT variant (SEQ ID NO: 4) and iPP co-immobilized on ChiralVision IB-HIS-2 at 2.5 and 0.02 wt. %, respectively, according to the procedure in Example 22.
  • the column bed was maintained at 40 °C via heated water jacket throughout the duration of the experiment.
  • the reaction solution was recirculated through the packed resin bed using a peristaltic pump with a flow rate of 10.2 column volumes per hour until the reaction had reached completion as determined by the HPLC method described in Example 4, Method A.
  • a Cytiva XK 16/40 column was packed with 20.3 g AP immobilized on ChiralVision IB- COV-7 at 2.0 wt. % according to the procedure in Example 23.
  • the column bed was maintained at 50 °C via heated water jacket throughout the duration of the experiment.
  • the reaction solution was pumped through the packed resin bed using a peristaltic pump at a flow rate of 6.8 column volumes per hour until the entire reaction solution had been pumped through the resin bed and collected in a separate vessel.
  • the level of 3’P-dephosphorylation was measured via HPLC as described in Example 4, Method A. Dephosphorylation of residual mUTP-3’P was measured by the HPLC method as described in Example 3.
  • the residual AP activity of the final reaction solution was measured as described in Example 6.
  • the residual C0CI2 concentration of the final reaction solution was measured at 0.42 mM C0CI2 according to the method described in Example 7 and was adjusted to 1.0 mM final C0CI2 concentration prior to moving on to the next cycle.
  • a Cytiva XK 16/40 column was packed with 20.3 g AP immobilized on ChiralVision IB- COV-7 at 2.0 wt. % according to the procedure in Example 23.
  • the column bed was maintained at 50 °C via heated water jacket throughout the duration of the experiment.
  • the reaction solution was pumped through the packed resin bed using a peristaltic pump at a flow rate of 6.8 column volumes per hour until the entire reaction solution had been pumped through the resin bed and collected in a separate vessel.
  • the level of 3’P-dephosphorylation was measured via HPLC as described in Example 4, Method A. Dephosphorylation of residual fCTP-3’P was measured by the HPLC method as described in Example 3.
  • a Cytiva XK 16/40 column was packed with 20.3 g AP immobilized on ChiralVision IB- COV-7 at 2.0 wt. % according to the procedure in Example 23.
  • the column bed was maintained at 50 °C via heated water jacket throughout the duration of the experiment.
  • the reaction solution was pumped through the packed resin bed using a peristaltic pump at a flow rate of 6.8 column volumes per hour until the entire reaction solution had been pumped through the resin bed and collected in a separate vessel.
  • the level of 3’P-dephosphorylation was measured via HPLC as described in Example 4, Method A. Dephosphorylation of residual mUTP-3’P was measured by the HPLC method as described in Example 4, Method A.
  • the residual AP activity of the final reaction solution was measured as described in Example 6.
  • the residual C0CI2 concentration of the final reaction solution was measured at 0.54 mM C0CI2 according to the method described in Example 7 and was adjusted to 1.0 mM final C0CI2 concentration prior to moving on to the next cycle.
  • a Cytiva XK 16/40 column was packed with 20.3 g AP immobilized on ChiralVision IB- COV-7 at 2.0 wt. % according to the procedure in Example 23.
  • the column bed was maintained at 50 °C via heated water jacket throughout the duration of the experiment.
  • the reaction solution was pumped through the packed resin bed using a peristaltic pump at a flow rate of 6.8 column volumes per hour until the entire reaction solution had been pumped through the resin bed and collected in a separate vessel.
  • the level of 3’P-dephosphorylation was measured via HPLC as described in Example 4, Method A.. Dephosphorylation of residual fCTP-3’P was measured by the HPLC method as described in Example 3.
  • the residual AP activity of the final reaction solution was measured as described in Example 6.
  • the residual CoCL concentration of the final reaction solution was measured at 0.9 mM CoCL according to the method described in Example 7 and was adjusted to 2.0 mM final C0CI2 concentration prior to moving on to the next cycle.
  • a Cytiva XK 16/40 column was packed with 20.3 g AP immobilized on ChiralVision IB- COV-7 at 2.0 wt. % according to the procedure in Example 23.
  • the column bed was maintained at 50 °C via heated water jacket throughout the duration of the experiment.
  • the reaction solution was pumped through the packed resin bed using a peristaltic pump at a flow rate of 6.8 column volumes per hour until the entire reaction solution had been pumped through the resin bed and collected in a separate vessel.
  • the level of 3’P-dephosphorylation was measured via HPLC as described in Example 4, Method A. Dephosphorylation of residual mATP-3’P was measured by the HPLC method as described in Example 3.
  • a Cytiva XK 16/40 column was packed with 20.3 g AP immobilized on ChiralVision IB- COV-7 at 2.0 wt. % according to the procedure in Example 23.
  • the column bed was maintained at 50 °C via heated water jacket throughout the duration of the experiment.
  • the reaction solution was pumped through the packed resin bed using a peristaltic pump at a flow rate of 6.8 column volumes per hour until the entire reaction solution had been pumped through the resin bed and collected in a separate vessel.
  • the level of 3’P-dephosphorylation was measured via HPLC as described in Example 4, Method A. Dephosphorylation of residual mUTP-3’P was measured by the HPLC method as described in Example 3.

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Abstract

La présente invention concerne des procédés et des systèmes de synthèse sans matrice d'un oligonucléotide, et des procédés d'utilisation des systèmes tels que divulgués dans la description, pour la synthèse sans matrice d'un oligonucléotide.
PCT/US2025/024415 2024-04-12 2025-04-11 Systèmes et procédés de synthèse enzymatique d'un oligonucléotide Pending WO2025217610A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180362969A1 (en) * 2017-06-19 2018-12-20 Massachusetts Institute Of Technology Automated methods for scalable, parallelized enzymatic biopolymer synthesis and modification using microfluidic devices
US20210009994A1 (en) * 2018-12-13 2021-01-14 Dna Script Sas Direct oligonucleotide synthesis on cells and biomolecules
WO2023031333A1 (fr) * 2021-09-01 2023-03-09 Synhelix Support polymère soluble, son procédé de synthèse et ses utilisations

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180362969A1 (en) * 2017-06-19 2018-12-20 Massachusetts Institute Of Technology Automated methods for scalable, parallelized enzymatic biopolymer synthesis and modification using microfluidic devices
US20210009994A1 (en) * 2018-12-13 2021-01-14 Dna Script Sas Direct oligonucleotide synthesis on cells and biomolecules
WO2023031333A1 (fr) * 2021-09-01 2023-03-09 Synhelix Support polymère soluble, son procédé de synthèse et ses utilisations

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