[go: up one dir, main page]

WO2024264000A2 - Synthèse de novo indépendante d'un modèle par étapes de polynucléotides longs - Google Patents

Synthèse de novo indépendante d'un modèle par étapes de polynucléotides longs Download PDF

Info

Publication number
WO2024264000A2
WO2024264000A2 PCT/US2024/035137 US2024035137W WO2024264000A2 WO 2024264000 A2 WO2024264000 A2 WO 2024264000A2 US 2024035137 W US2024035137 W US 2024035137W WO 2024264000 A2 WO2024264000 A2 WO 2024264000A2
Authority
WO
WIPO (PCT)
Prior art keywords
nucleotide
group
polymerase
polynucleotides
linker
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/US2024/035137
Other languages
English (en)
Other versions
WO2024264000A3 (fr
Inventor
Sebastian PALLUK
Daniel ARLOW
Eric ESTRIN
Jared ELLEFSON
Aaron Feldman
Jeffrey George BERTRAM
Uwe Theo BORNSCHEUER
Nico Dennis FESSNER
Christoffel Petrus Stephanus Badenhorst
Seema MENGSHETTI
Ben RALISKI
Robert Nichols
Sebastian BARTHEL
Ronald T. Raines
Tuan Tran
Evan Foster
Joseph KOSCINSKI
Christopher HOFF
Margarita KHARITON
Julia Swavola
Urmi BHAUMIK
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.)
Ansa Biotechnologies Inc
Original Assignee
Ansa Biotechnologies 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 Ansa Biotechnologies Inc filed Critical Ansa Biotechnologies Inc
Publication of WO2024264000A2 publication Critical patent/WO2024264000A2/fr
Publication of WO2024264000A3 publication Critical patent/WO2024264000A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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

  • Polynucleotide synthesis includes creation of chains of nucleotides, which are building blocks of DNA and RNA.
  • Standard de novo DNA synthesis performed today is based on the nucleoside phosphoramidite method (generally referred to as “chemical synthesis”) in which a desired sequence is synthesized by stepwise coupling of blocked monomers. These reactions are performed in organic solvents using highly-reactive activated monomers, and the conditions cause side reactions that damage the growing chain, limiting the yield of full-length product.
  • the impurities produced can be difficult or impractical to separate from the desired oligonucleotide product, limiting the usefulness of the method for producing sequences longer than approximately 200 bases.
  • stepwise yield and length have still not significantly improved.
  • stepwise yield can start to stall after the synthesized polynucleotide has reached a certain length. Even if the stepwise yield remains consistent, for polynucleotides up to 500 or 1000 nucleotides in length, a very high stepwise yield is needed to achieve a reasonable number of perfectly synthesized long polynucleotides via de novo stepwise synthesis.
  • the present disclosure provides technologies (e.g., methods of synthesizing, polynucleotides, etc.) that represent advances and improvements in long polynucleotide synthesis.
  • the disclosure is based, in part, upon methods of synthesis for long polynucleotides, including improved methods that achieve not only a high stepwise yield and/or lower error rate than previous methods, but do so in successfully producing long polynucleotides (e.g., about 500 to about 1000 nucleotides or more in length).
  • the present disclosure provides a method of template-independent de novo synthesis of a long single-stranded polynucleotide, comprising providing a substrate comprising a plurality of free hydroxyl groups linked to the substrate and suitable for nucleotide coupling; coupling a nucleotide to the plurality of free hydroxyl groups, wherein the nucleotide is linked to a blocking group; removing said blocking group from said coupled nucleotides; repeating steps (b) and (c) according to a predetermined nucleotide sequence (e.g., a reference sequence) to yield a plurality of de novo synthesized polynucleotides at least 500 nucleotides in length, wherein each coupling has an observed error rate of less than 1% as compared to said predetermined nucleotide sequence.
  • a predetermined nucleotide sequence e.g., a reference sequence
  • the de novo synthesized long polynucleotides is at least 600, at least 700, at least 800, at least 900, or at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, or at least 2000 nucleotides in length.
  • the de novo synthesized long polynucleotides from 500 to 1000 nucleotides in length, from 500 to 1500 nucleotides in length, from 500 to 2000 nucleotides in length, or from 500 to 2500 nucleotides in length.
  • the observed error rate of each coupling is less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1% per cycle, less than 0.09% per cycle, less than 0.08% per cycle, less than 0.07% per cycle, less than 0.06% per cycle, less than 0.05% per cycle, less than 0.04% per cycle, less than 0.03% per cycle, less than 0.02% per cycle, or less than 0.01% per cycle.
  • said coupling step is performed in less than 120 seconds, less than 90 seconds, less than 60 seconds, less than 50 seconds, or less than 40 seconds, less than 30 seconds, less than 20 seconds, less than 15 seconds, less than 10 seconds, or less than 5 seconds.
  • said template-independent polynucleotide synthesis to yield said de novo synthesized polynucleotides is performed at a rate of at least 10 nucleotides per hour, at least 15 nucleotides per hour, at least 20 nucleotides per hour, at least 25 nucleotides per hour, at least 30 nucleotides per hour, at least 40 nucleotides per hour, at least 50 nucleotides per hour, or at least 60 nucleotides per hour.
  • said template-independent polynucleotide synthesis is performed at a rate of from 5 nucleotides per hour to 25 nucleotides per hour, from 10 nucleotides per hour to 30 nucleotides per hour, from 15 nucleotides per hour to 45 nucleotides per hour, or from 20 nucleotides per hour to 60 nucleotides per hour.
  • said plurality of de novo synthesized polynucleotides on the substrate comprises at least 10 polynucleotides, at least 20 polynucleotides, at least 50 polynucleotides, at least 100 polynucleotides, at least 200 polynucleotides, at least 500 polynucleotides, at least 1,000 polynucleotides, at least 10,000 polynucleotides, at least 10 x 10 5 polynucleotides, at least 10 x 10 6 polynucleotides, at least 10 x 10 7 polynucleotides, at least 10 x 10 8 polynucleotides, at least 10 x 10 9 polynucleotides, at least 10 x 10 10 polynucleotides, at least 10 x 10 11 polynucleotides, at least 10 x 10 12 polynucleotides, at least 10 x 10 13 polynucleotides, or at
  • the free hydroxyl groups are at the end of a plurality of starter oligonucleotides or growing polynucleotides attached to the substrate.
  • the starter oligonucleotides comprise a single-stranded region at the 3’ end.
  • the starter oligonucleotide is hybridized to an oligonucleotide bound to the substrate.
  • the starter oligonucleotide is covalently linked to the substrate.
  • said nucleotide coupling is performed enzymatically.
  • said nucleotide coupling is catalyzed by a polymerase.
  • said polymerase is a template-independent polymerase.
  • said template-independent polymerase is covalently linked to said nucleotide.
  • said template-independent polymerase is Terminal deoxynucleotidyl Transferase (TdT), or a variant thereof.
  • said polymerase is an RNA polymerase.
  • said blocking group is a template-independent polymerase linked to said nucleotide.
  • said blocking group comprises cleaving a linker attaching said nucleotide to said template-independent polymerase.
  • said blocking group is a 3'-O-blocking group.
  • removing said blocking group comprises removing said 3'-O- blocking group from said nucleotide to leave a free 3' hydroxyl group.
  • the blocking group is a 2' or 3' modification of the nucleotide.
  • the 2' modification is selected from the group consisting of - H, -OH, -F, -OMe, -N3, -NH2, and -Ara.
  • the 3' modification is selected from the group consisting of — H, -OH, -OCH2N3, -ONH2 and -Oallyl.
  • the blocking group is a reversible terminator.
  • the predetermined sequence has a GC content of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.
  • the predetermined sequence has an AT content of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.
  • said coupling is performed in the presence of phosphatase.
  • the phosphatase is an inorganic pyrophosphatase.
  • the nucleotide linked to the blocking group is a nucleotidepolymerase conjugate and the conjugate has been treated with phosphatase.
  • said coupling is performed in the presence of a divalent cation having a total concentration of divalent cations present in the reaction volume of said coupling no greater than about 500 pM.
  • the total concentration of divalent cations present in the reaction volume is no greater than about 250 pM, about 125 pM or about 50 pM.
  • the divalent cation present at the highest concentration in the reaction volume is cobalt (Co2+) or zinc (Zn2+).
  • the at least one divalent cation is selected from Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Fe2+, Ni2+, Cu2+, and Zn2+, or a combination thereof.
  • the coupling reaction is performed in the absence of Mg2+.
  • said nucleotide comprises one or more modifications to a hydrogen binding N or O on the nucleobase.
  • said coupled nucleotide comprises one or more alkylated nucleobases after removal of said blocking group.
  • the method further comprises contacting said de novo synthesized polynucleotides with an alkyl transferase.
  • said alkyl transferase is from EC 2.1.1.63.
  • said alkyl transferase is selected from an alkyl transferase listed in Table 1 or Table 2.
  • said alkyl transferase is O6-alkylguanine DNA alkyltransferase.
  • said alkyl transferase is AlkB.
  • R1 is C1-4 alkyl.
  • R1 is selected from the group consisting of methyl, ethyl, n- propyl, and n-butyl.
  • R1 is selected from the group consisting of: .
  • R1a is -OR1b.
  • R1b is hydrogen.
  • the alkylated nucleobase in the polynucleotide is selected from the group consisting of:
  • said coupled nucleotide comprises one or more modifications to a base-pairing nitrogen or oxygen on the nucleobase after removal of said blocking group.
  • said coupled nucleotide is represented by (B): (B) wherein R is a ribose polyphosphate or deoxyribose polyphosphate; Y is a nucleobase; L- R1 is a protecting group; wherein L is attached to a base-pairing nitrogen or oxygen of the nucleobase; and wherein R1 is selected from the group consisting of hydrogen, -OH, - N(Rb )2, and -SH, wherein each Rb is independently hydrogen or optionally substituted C1-6 alkyl.
  • L is -Z-L1-L2-;
  • Z is selected from the group consisting of a bond, -C(O)-, -C(O)CH2-, -C(O)C(RL)2-, -C(O)CH(RL)-, -C(O)O-, and -C(O)N(H)-;
  • L1 is selected from the group consisting of a bond, , each RL is independently selected from the group consisting of halogen, hydroxyl, oxo, and optionally substituted C1-C3 alkyl, wherein 2 instances of R1 are optionally taken together with the intervening atom(s) to form a 3-6 membered carbocyclyl ring;
  • L2 is selected from the group consisting of a bond, an optionally substituted C1-12 alkylene chain, C4-C20 polyethylene glycol, an optionally substituted C2-12 alkenylene chain, and an optionally substituted C2-12 al
  • Z is a bond when L is attached to a base-pairing oxygen of the nucleobase.
  • Z is selected from the group consisting of -C(O)-, -C(O)CH2- , -C(O)C(RL)2-, -C(O)CH(RL)-, -C(O)O-, and -C(O)N(H)-when L is attached to a base- pairing nitrogen of the nucleobase.
  • L1 is , wherein L1 is optionally substituted with 1-4 instances of RL; n is 1 or 2; and W is selected from the group consisting of -O-, -S-, - S(O)2-, and -N(Rb)-. [0064] In some embodiments, L1 is selected from the group consisting of , wherein L1 is optionally substituted with 1-4 instances of RL. [0065] In some embodiments, each RL is independently optionally substituted C1-C3 alkyl, wherein 2 instances of R1 are optionally taken together with the intervening atom(s) to form a 3-6 membered carbocyclyl ring.
  • RL is optionally substituted methyl.
  • L1 is selected from the group consisting of .
  • Z is -C(O)O-.
  • Z is -C(O)N(H)-.
  • Z is a bond.
  • Z is -C(O)-.
  • -Z-L1-L2-R1 is selected from the group consisting of [0074]
  • L2 is an optionally substituted C1-12 alkylene chain, wherein 1-6 methylene units are optionally and independently replaced with -O-, -N(Rb)-, -C(O)-, -S- , -S(O)-, -S(O)2-, or phenylene.
  • L2 is an optionally substituted C1-12 alkylene chain, wherein 1-6 methylene units are optionally and independently replaced with -O-.
  • L2 is an optionally substituted C2-6 alkylene chain, wherein 1-3 methylene units are optionally and independently replaced with -O-.
  • -Z-L1-L2-R1 is selected from the group consisting of [0078]
  • said coupling comprises dipping said substrate into a solution comprising said nucleotide and a template-independent polymerase.
  • the blocking group is a polymerase, and wherein the polymerase is linked to the nucleotide via a cleavable linker.
  • the cleavable linker comprises an amino acid ester.
  • the amino acid ester is attached to an amino acid.
  • the amine group of the amino acid ester is bound to the amino acid.
  • the cleavable linker comprises a peptide of at least 2, at least 3, at least 4, or at least 5 amino acids bound to the amine group of the amino acid ester.
  • the amino acid or amino acids is selected from the group consisting of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • the amino acid is glycine or the amino acids comprise glycine.
  • the amino acid is a non-naturally occurring amino acid or the amino acids comprise a non-naturally occurring amino acid.
  • the cleavable linker is bound to the alpha-phosphate, sugar, or nucleobase of the nucleotide.
  • the amino acid ester is represented by: ; wherein R1 and R1' are each independently selected from hydrogen and an optionally substituted C1-6 alkyl, or are optionally taken together with the atom on which they are attached to form an optionally substituted C3-C7 carbocyclic ring.
  • the amino acid ester is represented by a compound selected from the group consisting of: [0090]
  • the linker comprises the structure: wherein R1 and R1' are each independently selected from hydrogen and an optionally substituted C1-6 alkyl or are optionally taken together with the atom on which they are attached to form an optionally substituted C3-C7 carbocyclic ring; each R2 is an optionally substituted group independently selected from the group consisting of hydrogen, C1-6 alkyl, phenyl, C1-C6 carbocyclic ring and 3-7 heterocyclic ring; each R3 is hydrogen or optionally substituted C1-6 alkyl; and n is 1, 2, 3, 4 or 5.
  • R3 is hydrogen.
  • R2 is hydrogen.
  • R2 is selected from the group consisting of hydrogen, -Me, - iso-Pr, -sec-butyl, iso-butyl, -CH2Ph, -CH2OH, -CH2SH, -CH2CH2SCH3, -CH2COOH, - CH2CH2COOH, -CH2CONH2, -CH2CH2CONH2, -CH2CH2, -CH2CH2NH2, [0094] In some embodiments, n is 1.
  • R1 and R1' are taken together to form an optionally substituted C3-C7 carbocyclic ring.
  • R1 and R1' are taken together to form an optionally substituted C3 carbocyclic ring.
  • the linker comprises the structure: .
  • the nucleotide linked to the polymerase comprises the structure: Nuc—L1—L2—L3—Pol, wherein: Nuc is the nucleotide; Pol is the polymerase; L1 is a first portion of the linker connecting the nucleotide to L2; L2 is a second portion of the linker represented by: wherein R1 and R1' are each independently selected from an optionally substituted C1-6 alkyl, a halogen, or are optionally taken together with the atom on which they are attached to form an optionally substituted C3- C7 carbocyclic ring; each R2 is an optionally substituted group independently selected from the group consisting of hydrogen, C1-6 alkyl, phenyl, C1-C6 carbocyclic ring and 3-7 heterocyclic ring; each R3 is hydrogen or optionally substituted C1-6 alkyl; n is 0, 1, 2, 3, 4 or 5; wherein * indicates the attachment point of L2 to L1; and ** indicates
  • L1 comprises: or TMS ; each Ra is independently selected from the group consisting of halogen, hydroxyl, cyano, optionally substituted C1-6 alkyl, and optionally substituted C1-6 alkoxy.
  • L2 comprises an amino acid ester selected from the group consisting of: [0102] In some embodiments, L2 is represented by: .
  • L1 is bound to the nucleobase of the nucleotide.
  • L1 is bound to the nucleobase at an oxygen or nitrogen involved in base pairing.
  • the nucleobase is selected from the group consisting of:
  • L1 is bound to the sugar of the nucleotide. [0107] In some embodiments, L1 is bound to a phosphate of the nucleotide. [0108] In some embodiments, the phosphate is the alpha phosphate. [0109] In some embodiments, the nucleotide is a ribonucleotide polyphosphate or a deoxyribonucleotide polyphosphate. [0110] In some embodiments, the nucleotide is selected from the group consisting of: adenine, guanine, cytosine, uracil, and thymine. [0111] In some embodiments, the polymerase is a template-independent polymerase.
  • the polymerase is TdT.
  • the linker is capable of being cleaved by a protease comprising esterase activity.
  • the linker is capable of being cleaved by Proteinase K.
  • said linker is capable of being cleaved at the ester group on L2, leaving a compound represented by Nuc-L1-OH after said cleavage.
  • the present disclosure provides substrate comprising a plurality of attached polynucleotides at least 500 nucleotides in length, wherein said plurality of polynucleotides are characterized by sequences generated by a stepwise template- independent polynucleotide synthesis having an observed error rate of less than 1% per cycle as compared to a predetermined nucleotide sequence.
  • the polynucleotides are at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, or at least 2000 nucleotides in length.
  • the polynucleotides are from 500 to 1000 nucleotides in length, from 500 to 1500 nucleotides in length, from 500 to 2000 nucleotides in length, or from 500 to 2500 nucleotides in length.
  • the observed error rate is less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1% per cycle, less than 0.09% per cycle, less than 0.08% per cycle, less than 0.07% per cycle, less than 0.06% per cycle, less than 0.05% per cycle, less than 0.04% per cycle, less than 0.03% per cycle, less than 0.02% per cycle, or less than 0.01% per cycle as compared to said predetermined sequence.
  • the observed error rate is greater than 0.001%, greater than 0.002%, greater than 0.005%, greater than 0.01%, greater than 0.02%, greater than 0.05%, or greater than 0.1% per cycle as compared to said predetermined sequence.
  • the plurality of attached polynucleotides on the substrate comprises at least 10 polynucleotides, at least 20 polynucleotides, at least 50 polynucleotides, at least 100 polynucleotides, at least 200 polynucleotides, at least 500 polynucleotides, at least 1,000 polynucleotides, at least 10,000 polynucleotides, at least 10 x 10 5 polynucleotides, at least 10 x 10 6 polynucleotides, at least 10 x 10 7 polynucleotides, at least 10 x 10 8 polynucleotides, at least 10 x 10 9 polynucleotides, at least 10 x 10 10 polynucleotides, at least 10 x 10 11 polynucleotides, at least 10 x 10 12 polynucleotides, at least 10 x 10 13 polynucleotides, or at least 10 x 10
  • the polynucleotides comprise one or more nucleotides comprising a modification to a hydrogen binding N or O on the nucleobase.
  • FIG. 1B depicts a scheme for two-step cyclic nucleic acid synthesis using TdT- dNTP conjugates comprising a TdT molecule site-specifically linked to a dNTP via a cleavable linker.
  • FIG. 2 depicts (i) typical enzymatic DNA synthesis performed with an enzyme and free nucleotides with 3’ blocking groups, which can be inhibited by the formation of secondary structure during synthesis, and (ii) a diagram of improved conjugate-based synthesis provided herein, including use of polymerase-nucleotide conjugates comprising the polymerase linked to a base pairing N or O atom of the nucleobase.
  • a scarred nucleotide comprising a portion of the linker (scar) at the N or O atom is retained in the polynucleotide, which inhibits secondary structure formation.
  • FIG. 3 depicts a scheme for nucleic acid synthesis using TdT-dNTP conjugates where the dNTP has an O-alkyl modification separate from its binding site to the linker. Cyclic nucleic acid synthesis comprises cleavage of the linker to remove TdT from the added dNTP using a cleavage agent which leaves a scar. Once nucleic acid synthesis is complete, the O-alkyl group is removed from the nucleotide(s) using AGT. [0128] FIG.
  • FIG. 4 depicts a scheme for nucleic acid synthesis using TdT-dNTP conjugates where cleavage of the linker to remove TdT from the added dNTP using a cleavage agent results in a nucleotide comprising an O-alkyl scar from the cleavage.
  • the O-alkyl group is removed from the nucleotide(s) using AGT.
  • FIG. 5A shows exemplary intramolecular cyclization reactions for removal of a scar or protecting group comprising an unsubstituted and substituted alkyl group where the alkyl group is attached to an amide group linked to a nitrogen on the nucleobase.
  • FIG. 5A shows exemplary intramolecular cyclization reactions for removal of a scar or protecting group comprising an unsubstituted and substituted alkyl group where the alkyl group is attached to an amide group linked to a nitrogen on the nucleobase.
  • FIGS. 6A, 6B, and 6C are diagrams of exemplary unshielded nucleotides that could be present in a polymerase-nucleotide conjugate reagent.
  • FIG. 6A shows an exemplary template-independent polymerase with an exemplary unshielded nucleotide (e.g., a deoxynucleoside triphosphate or “dNTP”) tethered in the wrong position.
  • dNTP deoxynucleoside triphosphate
  • FIG. 6B shows an exemplary unfolded template-independent polymerase with an exemplary tethered nucleotide (e.g., dNTP).
  • FIG. 6C illustrates an exemplary “free” (or untethered) nucleotide (e.g., dNTP) as present in an exemplary conjugate reagent.
  • free dNTPs can be present in such polymerase-nucleotide conjugate reagent due to, e.g., cleavage of the linker between the nucleotide and the polymerase (e.g., due to instability) or, e.g., due to imperfect removal of free nucleotides from conjugates after conjugate synthesis.
  • FIG. 6D shows an exemplary polymerase-nucleotide conjugate comprising an exemplary shielded nucleotide (e.g., dNTP).
  • dNTP shielded nucleotide
  • the exemplary nucleotide is tethered in the catalytic site of a folded polymerase and is sterically hindered by the tethered polymerase from phosphatase cleavage at its 5′ phosphate.
  • FIG. 7 depicts a block diagram of a synthesis system that is suitable for exemplary embodiments.
  • FIG. 8 depicts a first illustrative configuration of the synthesis system.
  • FIG. 9 depicts a second illustrative configuration of the synthesis system.
  • FIG. 10 depicts an illustrative element and an illustrative well that are suitable for the exemplary embodiments.
  • FIG. 11 depicts illustrative cross-sections of element designs of exemplary embodiments.
  • FIG. 12 depicts a longitudinal view of illustrative element designs of exemplary embodiments.
  • FIG. 13A and 13B depict a floating elements design of an exemplary embodiment.
  • FIG. 14 depicts an illustrative reaction plate for use in exemplary embodiments. [0143] FIG.
  • FIG. 15 depicts a portion of an illustrative patterned surface for use in exemplary embodiments.
  • FIG. 16 depicts a flowchart of illustrative steps that may be performed in exemplary embodiments to synthesize polymers.
  • FIG. 17 depicts illustrative operations that may be performed in exemplary embodiments to perform hybridization of elements of an element array.
  • FIG. 18 depicts a flowchart of illustrative steps that may be performed in exemplary embodiments as part of the synthesis process.
  • FIG. 19 depicts a flowchart of illustrative steps that may be performed in exemplary embodiments to perform a cycle of the synthesis process.
  • FIG. 16 depicts a flowchart of illustrative steps that may be performed in exemplary embodiments to synthesize polymers.
  • FIG. 17 depicts illustrative operations that may be performed in exemplary embodiments to perform hybridization of elements of an element array.
  • FIG. 18 depicts a flowchart of illustr
  • FIG. 20 depicts an illustrative pattern of loading polymer extension solutions in wells of a reaction plate in exemplary embodiments.
  • FIG. 21 shows the results of extension reactions with TdT and (i) dGTP (left column) and (ii) O6-methyl dGTP (right column) performed for 30 seconds, 1 minute, 2 minutes, 4 minutes, or 8 minutes as described in Example 1.
  • the starter is unmodified T35 oligo (SEQ ID NO: 48).
  • the x-axis is the approximate oligo length in nucleotides and the y- axis is relative fluorescence of fluorescein at 517 nm.
  • FIG. 21 shows the results of extension reactions with TdT and (i) dGTP (left column) and (ii) O6-methyl dGTP (right column) performed for 30 seconds, 1 minute, 2 minutes, 4 minutes, or 8 minutes as described in Example 1.
  • the starter is unmodified T35 oligo (SEQ ID NO: 48).
  • FIG. 23 shows the results of a cyclic nucleotide synthesis reaction using TdT-dNTP conjugates using i) G nucleotides without alkylations in the O6 position (O7Et-G), ii) G nucleotides with alkylation in the O6 position only in two locations with the strongest predicted secondary structure, with the rest of the G nucleotides being without alkylations (O7Et-G & O6Bu-G), and iii) G nucleotides with alkylations in the O6 position (O6Bu-G), as described in Example 4. [0152] FIG.
  • FIG. 24 shows an agarose gel visualized with UV of the product of PCR amplification of a synthesized alkylated 50-mer that was treated or not treated with AGT for the time shown, as described in Example 5.
  • a non-alkylated positive control is also shown for reference.
  • FIG. 25 shows the results of a cyclic nucleotide synthesis reaction TdT-dNTP conjugates of a 50-mer polyG homopolymer (SEQ ID NO: 49) using G nucleotides with alkylations in the O6 position, as described in Example 6.
  • FIG. 26 shows the results of a cyclic nucleotide synthesis reaction TdT-dNTP conjugates of a 40-mer sequence with an expected 15 bp hairpin structure G nucleotides with alkylations in the O6 position, as described in Example 7.
  • FIG. 26 discloses SEQ ID NO: 35. [0155] FIG.
  • FIG. 27 shows a reaction scheme and to detect activity of human AGT on various O6-alkylated G nucleotides in a synthesized polynucleotide, and a gel showing the results of the assay for O6-methyl-G, O6-hydroxybutyl-G, O6-hydroxypropyl-G, N7-aminomethyl-O6- methyl-G, and O6-aminomethylbenzyl-G as described in Example 8. [0156] FIGs.
  • FIGs. 28A and 28B show the results of dealklylation reactions of oligonucleotide comprising 3’ G nucleotide with an O6-allyl modified G nucleotide at the 3’ end, with resulting oligonucleotides measured by capillary electrophoresis.
  • “Allyl-G” represents a control, untreated oligonucleotide with the O6-allyl modified G nucleotide at the 3’ end, while the remaining oligonucleotides were treated with the corresponding species of AGT as shown in FIGs. 28A and 28B. [0157] FIGs.
  • FIG. 29A and 29B show the result of polyG synthesis reactions performed without (“dGTP) and with an O6 alkyl modification at the 3’ end of a polyT starter oligo (FIG. 29A) and at the 3’ end of a polyC starter oligo (FIG. 29B).
  • FIG. 30 shows the results of incorporation of alkylated G and U nucleotides at the 3’ end of an oligonucleotide by a polymerase-nucleotide conjugate, followed by cleavage of the polymerase, leaving an alkyl scar on the G or U nucleotides.
  • FIG. 32A is an HPLC chromatogram showing the trace of the photocleavable dGTP nucleotide, the starting material for the photocleavage experiment. The X-axis is represented in minutes. The peak eluting at 5.693 minutes is representative of the intact photocleavable dATP nucleotide.
  • FIG. 32B is an HPLC chromatogram showing the trace of native dGTP nucleotide, the expected product of the photocleavage reaction. The X-axis is represented in minutes.
  • FIG. 32C is an HPLC chromatogram showing the trace of the reaction product of photocleavable dGTP after exposure to a 365 nm wavelength lamp for 120 minutes. The X- axis is represented in minutes.
  • FIG. 33A is an HPLC chromatogram showing the trace of the photocleavable dATP nucleotide, the starting material for the photocleavage experiment. The X-axis is represented in minutes. The peak eluting at 5.693 minutes is representative of the intact photocleavable dATP nucleotide.
  • FIG. 32C is an HPLC chromatogram showing the trace of the reaction product of photocleavable dGTP after exposure to a 365 nm wavelength lamp for 120 minutes. The X- axis is represented in minutes.
  • FIG. 33A is an HPLC chromatogram showing the trace of the photocleavable dATP nucleotide, the starting material for the photocleavage experiment. The X-axis is represented in minutes. The peak
  • FIG. 33B is an HPLC chromatogram showing the trace of the reaction product of photocleavable dATP after exposure to a 365 nm wavelength lamp for 120 minutes. The X- axis is represented in minutes. The peak eluting at 3.426 minutes is representative of the native dATP nucleotide.
  • FIG. 34A is a capillary electrophoresis pherogram showing UV exposed polynucleotide extension products following extension with a conjugate including TdT and a modified dGTP containing a photocleavable nitrobenzyl group.
  • FIG. 34A is a capillary electrophoresis pherogram showing UV exposed polynucleotide extension products following extension with a conjugate including TdT and a modified dGTP containing a photocleavable nitrobenzyl group.
  • FIG. 34B is a capillary electrophoresis pherogram showing polynucleotide extension products following extension with a conjugate including TdT and a modified dGTP containing a photocleavable nitrobenzyl group that were exposed to UV light.
  • FIG. 35 is a capillary electrophoresis pherogram showing polynucleotide extension products from conjugates with a linker attached to the N4 of cytosine and containing different removeable scars.
  • FIG. 36 is a capillary electrophoresis pherogram showing polynucleotide extension products from conjugates with a linker attached to the N6 of adenine and containing different removeable scars.
  • FIG. 35 is a capillary electrophoresis pherogram showing polynucleotide extension products from conjugates with a linker attached to the N6 of adenine and containing different removeable scars.
  • FIG. 37 is a capillary electrophoresis pherogram showing polynucleotide extension products from conjugates with a linker attached to the O4 of uracil and thymine and containing removeable scars.
  • the X-axis is represented in approximate oligonucleotide length in number of nucleotides, and the Y-axis is represented in relative fluorescence.
  • FIG. 38 is a capillary electrophoresis pherogram showing polynucleotide extension products from conjugates with a linker attached to the O6 of guanine and containing different removeable scars.
  • FIG. 39 is a capillary electrophoresis pherogram showing incubation of polynucleotide extension at pH 7 or pH 8 for various times following extension with a conjugate including TdT and a modified dGTP containing an eliminable sulfone group.
  • the first row contains a control ssDNA that serves as a marker for where a single natural guanine on the 3′ end of the starter oligo migrates.
  • Hashed vertical markers showing the migration of natural guanine and +1 sulfone-guanine are indicated for clarity.
  • FIG. 40 is a capillary electrophoresis pherogram showing the results of a single nucleotide extension of a starter oligonucleotide with a dGTP comprising a sulfide scar (thioether) (top), an oxidation or the reaction product to form a sulfone scar on the dGTP (middle), and exposure of the sulfone dGTP to sodium hydroxide to remove the O-linked scar from dGTP.
  • FIG. 41A is a capillary electrophoresis pherogram showing the results of treatment of an oligonucleotide comprising an N6 Carbamate Sulfide A scarred nucleotide with base (50 mM NaOH) to remove the scar and convert the scarred nucleotide to a native adenine.
  • FIG. 41B is a capillary electrophoresis pherogram showing the results of treatment of an oligonucleotide comprising a N4 Carbamate Sulfide C scarred nucleotide with base (50mM NaOH) to remove the scar and convert the scarred nucleotide to a native cytosine.
  • FIG. 41A is a capillary electrophoresis pherogram showing the results of treatment of an oligonucleotide comprising an N6 Carbamate Sulfide A scarred nucleotide with base (50 mM NaOH) to remove the scar and convert the scarred nucleo
  • FIG. 42 is a capillary electrophoresis pherogram showing the results of treatment of an oligonucleotide comprising an N6 Carbamate Ethyl A scarred nucleotide with base (50mM NaOH) to remove the scar and convert the scarred nucleotide to a native adenine.
  • FIG. 42 is a capillary electrophoresis pherogram showing the results of treatment of an oligonucleotide comprising an N6 Carbamate Ethyl A scarred nucleotide with base (50mM NaOH) to remove the scar and convert the scarred nucleotide to a native adenine.
  • oligonucleotide comprising a N6-linked scarred adenine
  • oligonucleotide after 30 minute treatment with triethylamine (TEA)
  • TAA triethylamine
  • the N6-linked scarred adenine are: N6 Carbamate Propyl A, N6 Carbamate Ethyl A, N6 Amide Propyl A, and N6 Amide Ethyl A.
  • FIG. 44 depicts an intramolecular cyclization reaction mechanism with kinetics impacted by the ring size for an N-linked carbamate scar or protecting group.
  • FIG. 45 shows the rate of the deprotection reaction of an N-linked scarred nucleotide incorporated into an oligonucleotide for the following scarred nucleotides: N6 Carbamate Ethyl A (large circles; Et-CO2-A), N6 Amide Propyl A (squares, Pr-CO-A), N4 Carbamate Ethyl C (triangles; Et-CO2-C) and N4 Carbamate (Methyl) Ethyl C (small circles, 2MeEt- CO2-C).
  • FIG. 46A shows the capillary electrophoresis analysis of uncontrolled oligonucleotide synthesis reactions using dG nucleotides to synthesize a G homopolymer on a 35T starter oligonucleotide (SEQ ID NO: 48).
  • dGTP represents a synthesis performed with unmodified nucleotides.
  • 06 Sulfone G and 06 Sulfide G represent syntheses performed with dGTP nucleotides modified to have a removable protecting group at the base pairing 06 atom of guanine. Oligo synthesis reactions were terminated at 30 seconds, 1 minute, 4 minutes, and 8 minutes to measure progress via capillary electrophoresis, as shown.
  • FIG. 46B shows the capillary electrophoresis analysis of uncontrolled oligonucleotide synthesis reactions using dG nucleotides to synthesize a G homopolymer on a 30C starter oligonucleotide (SEQ ID NO: 50).
  • dGTP represents a synthesis performed with unmodified nucleotides.
  • 06 Sulfone G and 06 Sulfide G represent syntheses performed with dGTP nucleotides modified to have a removable protecting group at the base pairing 06 atom of guanine. Oligo synthesis reactions were terminated at 30 seconds, 1 minute, 4 minutes, and 8 minutes to measure progress via capillary electrophoresis, as shown.
  • FIG. 47 shows the capillary electrophoresis analysis of uncontrolled oligonucleotide synthesis reactions using dA nucleotides to synthesize an A homopolymer on a 35T starter oligonucleotide (SEQ ID NO: 48).
  • dATP represents a synthesis performed with unmodified nucleotides.
  • N6 Carbamate Ethyl A and N6 Carbamate Sulfide A represent syntheses performed with dATP nucleotides modified to have a removable protecting group at the base pairing N6 atom of adenine.
  • Oligo synthesis reactions were terminated at 30 seconds, 1 minute, 4 minutes, and 8 minutes to measure progress via capillary electrophoresis, as shown.
  • FIG. 48 shows two amino acid ester dTTP analogs used for oligo synthesis and linker cleavage. One is based on a hydroxypropargyl scar (Linker 1) and the other on a smaller hydroxymethyl scar (Linker 2).
  • the two amino acid ester dTTP analogs (Linkers 1 and 2, FIG. 48; synthesized by Jena Bioscience) were attached to cysteine-reactive crosslinkers and conjugated to TdT with the final structure shown in FIG. 48. Also shown are the alcohol-scarred cleavage products after ester cleavage of the linker.
  • FIG. 49(A-C) shows a plot of the kinetics of conjugate addition to an unscarred oligo (FIG. 49(A and B)) and to a hydroxymethyl scarred oligo (FIG. 49-C)
  • FIG. 49-A Natural DNA primer exposed to a dTTP conjugate comprising an ester linkage for 1 second results in -35% extension yield.
  • FIG. 49-B The oligo synthesis reaction proceeds to completion, with linker cleavage yielding a primer with a hydroxymethyl scar on the last base.
  • FIG. 49-C Exposure of the scarred primer to the dTTP conjugate comprising an ester linkage for 1 second again results in -35% extension yield.
  • FIG. 50 shows the results of a primer extension by TdT- dTTP conjugates based on Linker 1 or Linker 2 as measured by a gel shift assay on SDS-PAGE.
  • An ssDNA primer was extended for 60s with 1) a Linker 1 conjugate, 2) a Linker 2 conjugate 3) a Linker 2 conjugate (replicates), 4) no conjugate.
  • T/P TdT/DNA primer complex.
  • P ssDNA primer.
  • FIG. 51 shows primer extension products as measured by capillary electrophoresis. Extension was performed by linker 2 conjugates stored overnight at the indicated pH, or in buffer only (negative control). Extension without insertion shows a peak at -58 nt. A peak indicating unwanted insertion (elongation products) is in some samples at -59 nt and indicates the presence of free dNTPs in the incubated conjugate.
  • FIG. 52 shows the results of an Enzymatic synthesis of lOOmer and 200mer dT oligos (SEQ ID NOs: 51 and 52, respectively) using the linker 2 dNTP conjugate as measured using capillary electrophoresis (part A).
  • An enlarged view of the product distributions of the 100 mers from enzymatic synthesis (top) and chemical synthesis (bottom) synthesis as observed via capillary electrophoresis is shown in part B.
  • FIG. 53 shows the results of an extension of an oligonucleotide using TdT-dATP, - dCTP, -dGTP, and -dTTP conjugates comprising linker 6 as measured by capillary electrophoresis (Panel A), and a cleavage time course of a TdT-dTTP conjugate comprising linker 6 incorporated into an oligonucleotide and cleaved via proteinase K for 30-240 seconds, as measured by capillary electrophoresis (Panel B).
  • FIG. 54 shows structures for a linker nucleotide comprising a glycine amino acid ester (Gly-OMe-U) and an ACC amino acid ester (ACC-OMe-U) and the product of ester instability of both linkers (HOMe-U) (top), and a comparison of the intact (Gly-OMe-U or ACC-OMe-U) and hydrolized (HOMe-U) product after 60 minutes of exposure to a temperature of 45°C.
  • Gly-OMe-U glycine amino acid ester
  • ACC-OMe-U ACC-OMe-U
  • HOMe-U the product of ester instability of both linkers
  • FIGs. 55A, 55B and 55C show a comparison of the linker cleavage efficiency of various TdT-nucleotide conjugates. Data shown is at the time point for 60 seconds of ProK treatment.
  • FIG. 56 shows a series of electropherographs characterizing the cleavage rate by Proteinase K (ProK) for illustrative linkers having an aminocyclopropyl carboxy ethyl group and either one (1XG) or two (2XG) glycines.
  • the cleavage reactions were quenched after 15 seconds (s), 30 s, 60 s, 4 minutes (m), 8 m, or 16 m.
  • FIG. 58 shows a plot of % ester hydrolysis for compounds 14-18 (ring expansion series linker nucleotides) after exposure to 50°C from 1 minute to 20 hours.
  • FIG. 59 shows the results of exposure to a temperature of 50°C for 1 hour, 4 hours, or overnight of an oligonucleotide extended with an Allyl G, ACC, AiB, AC4C, AC5C, or AC6C conjugate as measured by capillary electrophoresis to show proportion of intact and hydrolyzed products.
  • FIG. 60A shows results of an exemplary single nucleotide addition reaction onto an exemplary single- stranded DNA substrate using an A, C, T, or G polymerase conjugate in the presence (+Phos) or absence (-Phos) of phosphatase.
  • the resulting synthesized oligonucleotides were analyzed by capillary electrophoresis.
  • the x-axes show approximate nucleotide length of oligonucleotides and the y-axes indicate relative fluorescence at 517 nm. Reactions were terminated at the timepoints shown.
  • FIG. 60B shows an expanded view of the 21min 41s timepoint results from FIG.
  • FIG. 61 A shows graphical representations of results of capillary electrophoresis analysis of single nucleotide addition reactions onto a single- stranded DNA substrate using a T-polymerase conjugate in the presence of exemplary phosphatase variants from: B. taurus (Quick CIP, NEB), P. borealis (shrimp alkaline phosphatase, NEB), Antarctic bacterium TAB5 (Antarctic phosphatase, NEB), or E. coli (Takara Bio) phosphatase.
  • Synthesis reactions were performed at room temperature (24 °C). A control synthesis reaction was performed without phosphatase. Reactions were terminated at the timepoints shown.
  • the x- axes show relative electrophoretic migration of oligonucleotides (via approximate nucleotide length) and the y-axes indicate relative fluorescence at 517 nm.
  • FIG. 6 IB shows graphical representations of results of capillary electrophoresis analysis of an exemplary single nucleotide addition reaction onto a single- stranded DNA substrate using a T-polymerase conjugate in the presence of exemplary phosphatase variants: B. taurus (Quick CIP, NEB), P. borealis (shrimp alkaline phosphatase, NEB), Antarctic bacterium TAB5 (Antarctic phosphatase, NEB), or E. coli (Takara Bio) phosphatase.
  • the synthesis reaction was performed at 37°C (plus and minus phosphatases) and terminated after 30 minutes.
  • the arrow designates the expected size of +2 additions.
  • FIG. 62 shows graphical representations of results of an exemplary conjugate-based polynucleotide synthesis of an exemplary 50-mer polynucleotide , conducted in presence or absence of phosphatase, with resulting synthesized polynucleotides distinguished by size along the x-axis using a SeqStudio Genetic Analyzer. Peaks corresponding to the starter oligo and the correct 50-mer synthesis product are labeled.
  • FIGs. 63A-63D show a series of electropherograms showing results from analysis of products at different time points in an enzymatic polynucleotide extension reaction performed using a polymerase-nucleotide conjugate in a reaction buffer containing low cobalt acetate concentration (0.05 mM CoOAc) or a standard cobalt acetate concentration.
  • FIG. 63E is a plot showing the quantification and analysis of products in FIGs. 63A-63D, and the associated calculated rates of reaction (kobs).
  • FIGs. 64A-64L show a series of electropherograms showing results from analysis of products at different time points in an enzymatic polynucleotide extension reaction performed using a polymerase-nucleotide conjugate in a reaction buffer containing a range of cobalt acetate concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc).
  • FIG. 64M is a plot showing the quantification and analysis of products in FIGs. 64A-64L, and the associated calculated rates of reaction (kobs). [0201] FIGs.
  • FIG. 65A-65L show a series of electropherograms showing results from analysis of products at different time points in an enzymatic polynucleotide extension reaction performed using a polymerase-nucleotide conjugate in a reaction buffer containing a range of zinc acetate (ZnOAc) concentrations (0.05 mM ZnOAc, 0.125 mM ZnOAc, 0.25 mM ZnOAc, 0.75 mM ZnOAc, 1.25 mM ZnOAc, and 2.5 mM ZnOAc).
  • FIG. 65M is a plot showing the quantification and analysis of products in FIGs. 65A-65L, and the associated calculated rates of reaction (kobs).
  • FIGs. 66A-66L show a series of electropherograms showing results from analysis of products at different time points in an enzymatic polynucleotide extension reaction using a free polymerase and free nucleotide performed in a reaction buffer containing a range of cobalt acetate (CoOAc) concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc).
  • FIG. 66M is a plot showing the quantification and analysis of products in FIGs. 66A-66L.
  • FIG. 67 shows the percent of perfect polynucleotides at each step of synthesis of a 520 mer polynucleotide as measured by next generation sequencing.
  • FIG. 68 shows the percent of perfect polynucleotides at each step of synthesis of a 1005 mer polynucleotide as measured by next generation sequencing.
  • FIG. 69 shows charts characterizing the length and synthesis quality (stepwise yield) of oligonucleotides generated from the process described in Example 35.
  • FIG. 70 shows charts characterizing the length and synthesis quality (stepwise yield) of oligonucleotides greater than 1000 nucleotides in length and generated from the process described in Example 35.
  • FIG. 71 shows charts characterizing the length and synthesis quality of an ‘all5mer’ oligonucleotide sequence generated from the process described in Example 35.
  • compounds of the disclosure may contain “optionally substituted” moieties.
  • substituted whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent.
  • an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
  • Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds.
  • stable refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.
  • alkyl refers to a straight or branched full saturated hydrocarbon chain. Exemplary alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.
  • haloalkyl refers to a straight or branched alkyl group that is substituted with one or more halogen atoms.
  • compounds of the present disclosure may contain “optionally substituted” moieties.
  • substituted whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent.
  • an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
  • Combinations of substituents envisioned by this present disclosure are preferably those that result in the formation of stable or chemically feasible compounds.
  • Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; —(CH2)0-4R ⁇ ; —(CH2)0-4OR ⁇ ; —O(CH2)0-4R ⁇ , —O—(CH2)0-4C(O)OR ⁇ ; —(CH2)0-4CH(OR ⁇ )2; —(CH2)0-4SR ⁇ ; —(CH2)0-4Ph, which may be substituted with R ⁇ ; —(CH2)0-4O(CH2)0-1Ph which may be substituted with R ⁇ ; —CH ⁇ CHPh, which may be substituted with R ⁇ ; —(CH2)0-4O(CH2)0
  • Suitable monovalent substituents on R ⁇ are independently halogen, —(CH 2 ) 0-2 R ⁇ , -(haloR ⁇ ), —(CH 2 ) 0-2 OH, —(CH 2 ) 0-2 OR ⁇ , —(CH 2 ) 0-2 CH(OR ⁇ ) 2 ; — O(haloR ⁇ ), —CN, —N 3 , —(CH 2 ) 0-2 C(O)R ⁇ , —(CH 2 ) 0-2 C(O)OH, —(CH 2 ) 0-2 C(O)OR ⁇ , — (CH2)0-2SR ⁇ , —(CH2)0-2SH, —(CH2)0-2NH2, —(CH2)0-2NHR ⁇ , —(CH2)0-2NR ⁇ 2, —NO2, —
  • Suitable divalent substituents on a saturated carbon atom of R ⁇ include ⁇ O and ⁇ S.
  • Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: ⁇ O, ⁇ S, ⁇ NNR*2, ⁇ NNHC(O)R*, ⁇ NNHC(O)OR*, ⁇ NNHS(O)2R*, ⁇ NR*, ⁇ NOR*, —O(C(R*2))2-3O—, or —S(C(R*2))2- 3 S—, wherein each independent occurrence of R* is selected from hydrogen, C 1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: —O(CR* 2 ) 2-3 O—, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable substituents on the aliphatic group of R* include halogen, —R ⁇ , -(haloR ⁇ ), —OH, —OR ⁇ , —O(haloR ⁇ ), —CN, —C(O)OH, —C(O)OR ⁇ , —NH 2 , —NHR ⁇ , —NR ⁇ 2 , or —NO2, wherein each R ⁇ is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include —R ⁇ , —NR ⁇ 2, —C(O)R ⁇ , —C(O)OR ⁇ , —C(O)C(O)R ⁇ , —C(O)CH2C(O)R ⁇ , — S(O) 2 R ⁇ , —S(O) 2 NR ⁇ 2 , —C(S)NR ⁇ 2 , —C(NH)NR ⁇ 2 , or —N(R ⁇ )S(O) 2 R ⁇ ; wherein each R ⁇ is independently hydrogen, C1-6 aliphatic which may be substituted as defined below, unsubstituted —OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrence
  • Suitable substituents on the aliphatic group of R ⁇ are independently halogen, —R ⁇ , - (haloR ⁇ ), —OH, —OR ⁇ , —O(haloR ⁇ ), —CN, —C(O)OH, —C(O)OR ⁇ , —NH2, —NHR ⁇ , — NR ⁇ 2 , or —NO 2 , wherein each R ⁇ is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, —CH2Ph, — O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • compounds described herein may also comprise one or more isotopic substitutions.
  • hydrogen may be 2 H (D or deuterium) or 3 H (T or tritium); carbon may be for example 13 C or 14 C; oxygen may be for example 18 O; nitrogen may be, for example, 15 N, and the like.
  • a particular isotope (e.g., 3 H, 13 C, 14 C, 18 O, or 15 N) can represent at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% of the total isotopic abundance of an element that occupies a specific site of the compound.
  • the terms “about” and “approximately” refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system.
  • “about” or “approximately” can mean within one or more than one standard deviation per the practice in the art.
  • “about” or “approximately” can mean a range of up to 10% (i.e., ⁇ 10%) or more depending on the limitations of the measurement system.
  • about 5 mg can include any number between 4.5 mg and 5.5 mg.
  • the terms can mean up to an order of magnitude or up to 5-fold of a value.
  • the meaning of “about” or “approximately” should be assumed to be within an acceptable error range for that particular value or composition.
  • the ranges and/or subranges can include the endpoints of the ranges and/or subranges.
  • Nucleic acids include recombinant and chemically- synthesized forms. Nucleic acids can be isolated. Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids (PNA) and non-naturally occurring nucleotide analogs), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids comprise polymers of nucleotides, where the nucleotides include natural or non-natural bases and/or sugars.
  • PNA peptide nucleic acids
  • Nucleic acids comprise naturally-occurring internucleosidic linkages, for example phosphodiester linkages. Nucleic acids can lack a phosphate group. Nucleic acids comprise non-natural internucleoside linkages, including phosphorothioate, phosphorothiolate, or peptide nucleic acid (PNA) linkages. In some embodiments, nucleic acids comprise a one type of polynucleotides or a mixture of two or more different types of polynucleotides [0223]
  • the term “operably linked” and “operably joined” or related terms as used herein refers to juxtaposition of components. The juxtapositioned components can be linked together covalently.
  • two nucleic acid components can be enzymatically ligated together where the linkage that joins together the two components comprises phosphodiester linkage.
  • a first and second nucleic acid component can be linked together, where the first nucleic acid component can confer a function on a second nucleic acid component.
  • linkage between a primer binding sequence and a sequence of interest forms a nucleic acid library molecule having a portion that can bind to a primer.
  • a transgene e.g., a nucleic acid encoding a polypeptide or a nucleic acid sequence of interest
  • a transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects expression of the transgene.
  • the vector comprises at least one host cell regulatory sequence, including a promoter sequence, enhancer, transcription and/or translation initiation sequence, transcription and/or translation termination sequence, polypeptide secretion signal sequences, and the like.
  • the host cell regulatory sequence controls expression of the level, timing and/or location of the transgene.
  • the procedure can include but are not limited to: nucleotide binding; nucleotide incorporation; de-blocking (e.g., removal of chain-terminating moiety); washing; removing; flowing; detecting; imaging and/or identifying.
  • linkage can comprise, for example, covalent, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, or affinity bonding, bonds or associations involving van der Waals forces, mechanical bonding, and the like.
  • such linkage occurs intramolecularly, for example linking together the ends of a single-stranded or double-stranded linear nucleic acid molecule to form a circular molecule.
  • such linkage can occur between a combination of different molecules, or between a molecule and a non-molecule, including but not limited to: linkage between a nucleic acid molecule and a solid surface; linkage between a protein and a detectable reporter moiety; linkage between a nucleotide and detectable reporter moiety; and the like.
  • linkages can be found, for example, in Hermanson, G., “Bioconjugate Techniques”, Second Edition (2008); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences” London: Macmillan (1998); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998).
  • the terms “extend”, “extending”, “extension” and other variants refers to incorporation of one or more nucleotides into a nucleic acid molecule (i.e. nucleotide coupling).
  • Nucleotide incorporation comprises polymerization of one or more nucleotides into the terminal 3′ OH end of a nucleic acid strand (e.g., a nucleic acid primer), resulting in extension of the nucleic acid strand (e.g., starter oligo). Nucleotide incorporation can be conducted with natural nucleotides and/or nucleotide analogs.
  • cleavable linker or “cleavable moiety” as used herein refers to a divalent or monovalent, respectively, moiety which is capable of being separated (e.g., detached, split, disconnected, hydrolyzed, a stable bond within the moiety is broken) into distinct entities.
  • a cleavable linker is cleavable (e.g., specifically cleavable) in response to external stimuli (e.g., enzymes, nucleophilic/basic reagents, reducing agents, photo-irradiation, electrophilic/acidic reagents, organometallic and metal reagents, or oxidizing reagents).
  • external stimuli e.g., enzymes, nucleophilic/basic reagents, reducing agents, photo-irradiation, electrophilic/acidic reagents, organometallic and metal reagents, or oxidizing reagents.
  • Cleavable linkers may be, by way of non-limiting example, electrophilically cleavable linkers, nucleophilically cleavable linkers, photocleavable linkers, cleavable under reductive conditions (for example disulfide or azide containing linkers), oxidative conditions, cleavable via use of safety-catch linkers and cleavable by elimination mechanisms.
  • electrophilically cleavable linkers nucleophilically cleavable linkers, photocleavable linkers, cleavable under reductive conditions (for example disulfide or azide containing linkers), oxidative conditions, cleavable via use of safety-catch linkers and cleavable by elimination mechanisms.
  • the cleavable linker is cleaved by contacting the cleavable linker with a cleaving agent (e.g., a reducing agent). In embodiments, the cleaving agent is... [0229]
  • a cleaving agent e.g., a reducing agent
  • the cleaving agent is...
  • the term “polymerase-compatible cleavable moiety” and “polymerase-compatible cleavable linker” as used herein refers to a cleavable moiety or cleavable linker which does not interfere with the function of a polymerase (e.g., a DNA polymerase or modified DNA polymerase, in incorporating the nucleotide, to which the polymerase-compatible cleavable moiety is attached, to the 3′ end of the newly formed nucleotide strand).
  • a polymerase e.g., a DNA polymerase or modified DNA polymerase
  • polymerase-compatible cleavable moiety does not decrease the function of a polymerase relative to the absence of the polymerase- compatible cleavable moiety.
  • polymerase-compatible cleavable moiety does not negatively affect DNA polymerase recognition.
  • the polymerase- compatible cleavable moiety does not negatively affect (e.g., limit) the read length of the DNA polymerase.
  • nucleotide refers to a molecule comprising a nucleoside and one or more phosphate groups.
  • a “nucleoside” refers to a molecule comprising a nucleobase (e.g., adenine, thymine, cytosine, guanine, or uracil) and a five carbon sugar (e.g., ribose or 2'-deoxyribose).
  • Exemplary nucleotides can be or comprise, without limitation, a nucleoside monophosphate, a nucleoside diphosphate, a nucleoside triphosphate, a nucleoside tetraphosphate, a nucleoside pentaphosphate, or a nucleoside hexaphosphate.
  • TdT and TdT variants can, in some embodiments, incorporate any nucleoside polyphosphate, including nucleotide analogs comprising modifications to the nucleobase.
  • nucleoside polyphosphate is a “nucleotide” and may be called a “nucleotide polyphosphate.”
  • a “nucleotide triphosphate” and a “nucleoside triphosphate” both refer to a nucleotide comprising a nucleobase, a sugar, and a polyphosphate consisting of three linked phosphate groups.
  • “non-termination” or “insertion” occurs when more than one nucleotide is added during a single step of a cyclic nucleotide extension.
  • phosphatase refers to an enzyme capable of removing the 5′ phosphate of a nucleotide, especially a nucleotide that is unshielded as part of an improperly formed conjugate or is not tethered to a polymerase.
  • phosphatase is meant to also include all phosphatase enzymes, engineered enzymes having phosphatase activity, or a functional fragment thereof, that is capable of removing one or more phosphate group(s) from a nucleotide.
  • a phosphatase can also refer to any biomolecule (e.g., a polypeptide or ribozyme) capable of removing one or more phosphate group(s) from a nucleotide, including an engineered enzyme having phosphatase activity or functional fragments thereof [0234]
  • the term “blocked nucleotide” or “shielded nucleotide” refers to a nucleotide that is sterically hindered by a tethered polymerase (or other entity or component such as, e.g., a blocking group) from a phosphatase capable of removing its 5′ phosphate.
  • such nucleotides are likely to inhibit subsequent nucleotide additions after having been added to an oligonucleotide and before removal of said tethered polymerase.
  • the term “unblocked nucleotide” or “unshielded nucleotide” refers to a nucleotide that is not sterically hindered by a tethered polymerase (or other entity or component such as, e.g., a blocking group) from a phosphatase capable of removing its 5′ phosphate.
  • an unshielded nucleotide may be tethered to a polymerase, such as in a misfolded polymerase or tethered at an incorrect position.
  • An unshielded nucleotide may be untethered (or free) from a polymerase. Unshielded nucleotides that have not been exposed to phosphatase are more likely to be erroneously added to a polynucleotide as an insertion after a shielded nucleotide has been properly added.
  • reagents, systems, and methods for stepwise polynucleotide synthesis that mitigate common errors during a synthesis reaction, such as an unwanted nucleotide insertion (e.g., due to addition of an incompletely blocked nucleotide to a growing polynucleotide strand, or premature removal of a blocking group during an extension reaction), or an unwanted nucleotide deletion (e.g., due to an incomplete reaction, such as failure to add a nucleotide during an extension reaction, or failure to remove a blocking group during a nucleotide deblocking reaction.).
  • an unwanted nucleotide insertion e.g., due to addition of an incompletely blocked nucleotide to a growing polynucleotide strand, or premature removal of a blocking group during an extension reaction
  • an unwanted nucleotide deletion e.g., due to an incomplete reaction, such as failure to add a nucleotide during an extension reaction, or failure to remove a blocking group during
  • modified nucleotides that inhibit secondary structure formation during enzymatic polynucleotide synthesis. Such secondary structure formation can inhibit extension of a growing polynucleotide by certain polymerases, such as TdT.
  • optimized reagent and cofactor conditions for improved enzymatic polynucleotide synthesis activity. Such improved reagents and methods inhibit deletions due to incomplete extensions, thereby improving stepwise yields of long polynucleotide synthesis.
  • reagents to inhibit unwanted insertions such as phosphatase treatment of nucleotide-linker conjugates, which inhibits activity of unblocked nucleotides during the extension step.
  • improved linkers between blocking groups e.g., TdT
  • the nucleotide that are stable during the extension step (to inhibit unwanted insertions), but quickly and completely cleave during the blocking group removal step (to inhibit unwanted deletions).
  • improved systems for oligonucleotide synthesis such as a synthesis surface that is dipped into the appropriate pre- prepared extension, blocking group removal, and wash buffers to improve synthesis speed and reagent delivery. Such systems can also act to improve overall stepwise yield and reaction cycle speed.
  • methods of de novo template- independent synthesis of a polynucleotide at least 500 bp in length with an observed error rate of less than 1% as compared to a predetermined sequence are provided herein.
  • the de novo synthesized long polynucleotides are at least 600, at least 700, at least 800, at least 900, or at least 1000 nucleotides in length. In some embodiments, the de novo synthesized long polynucleotides are from 500 to 1000 nucleotides in length. [0245] In some embodiments, the observed error rate of each coupling during de novo template-independent synthesis of a long polynucleotide is less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% per cycle.
  • the observed stepwise yield of a de novo template- independent synthesis of a long polynucleotide is greater than 99%, greater than 99.1%, greater than 99.2%, greater than 99.3%, greater than 99.4%, greater than 99.5%, greater than 99.6%, greater than 99.7%, greater than 99.8%, or greater than 99.9%. In some embodiments, the observed stepwise yield of a de novo template-independent synthesis of a long polynucleotide is from 99% to 99.9%.
  • each nucleotide coupling step of a de novo template- independent synthesis of a long polynucleotide is performed in less than 120 seconds, less than 90 seconds, less than 60 seconds, less than 50 seconds, or less than 40 seconds. In some embodiments, each nucleotide coupling step of a de novo template-independent synthesis of a long polynucleotide is performed at a rate of at least 10 nucleotides per hour, at least 15 nucleotides per hour, or at least 20 nucleotides per hour.
  • the nucleotide coupling to yield said de novo synthesized polynucleotides is performed at a rate of from 5 nucleotides per hour to 25 nucleotides per hour.
  • the de novo template-independent polynucleotide synthesis generates at least 10, at least 20, at least 50, at least 100, at least 500, at least 1000, at least 2000, or at least 5000 copies of each sequence.
  • the overall error rate or error rates for individual types of errors such as deletions, insertions, or substitutions for each oligonucleotide synthesized on the substrate, for at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or more of the oligonucleotides synthesized on the substrate, or for the substrate average may be at most or at most about 1:100, 1:500, 1:1000, 1:10000, 1:20000, 1:30000, 1:40000, 1:50000, 1:60000, 1:70000, 1:80000, 1:90000, 1:1000000, or less.
  • the overall error rate or error rates for individual types of errors such as deletions, insertions, or substitutions for each oligonucleotide synthesized on the substrate, for at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or more of the oligonucleotides synthesized on the substrate, or the substrate average may fall between 1:100 and 1:10000, 1:500 and 1:30000.
  • the overall error rate or error rates for individual types of errors such as deletions, insertions, or substitutions for each oligonucleotide synthesized on the substrate, for at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or more of the oligonucleotides synthesized on the substrate, or the substrate average may fall between any of these values, for example 1:500 and 1:10000.
  • Desired predetermined sequences may be supplied by any method, typically by a user, e.g., a user entering data using a computerized system.
  • synthesized nucleic acids are compared against these predetermined sequences in some cases by sequencing at least a portion of the synthesized nucleic acids, e.g., using next-generation sequencing methods.
  • the polynucleotides can be released from the substrate by a variety of suitable methods as described in further details elsewhere herein and known in the art, for example by enzymatic cleavage, as is well known in that art.
  • Examples of such enzymatic cleavage include, but are not limited to, the use of restriction enzymes such as MIyI, or other enzymes or combinations of enzymes capable of cleaving single or double-stranded DNA such as, but not limited to, Uracil DNA glycosylase (UDG) and DNA Endonuclease IV.
  • Other methods of cleavage known in the art may also be advantageously employed in the present disclosure, including, but not limited to, chemical (base labile) cleavage of DNA molecules or optical (photolabile) cleavage from the surface.
  • PCR or other amplification reactions can also be employed to generate building material for gene synthesis by copying the oligonucleotides while they are still anchored to the substrate.
  • Enzymatic Polynucleotide Synthesis [0252]
  • a method of polynucleotide synthesis to generate polynucleotides of desired length and sequence according to the embodiments described herein.
  • the steps are performed by dipping a reaction surface comprising a bound synthesis initiator (which may also include previously added nucleotides) into a contained solution comprising the desired reagents.
  • the method is amenable to multiplexed polynucleotide synthesis, such that a plurality of elements having an end with the reaction surface and bound synthesis initiator may be used and simultaneously dipped into a plurality of contained liquid reagents (such as in wells or droplets on a surface) aligned with the reaction surfaces.
  • the contained liquid reagent comprise a polymer extension solution with a nucleotide of a specific identity and polymerase capable of adding the nucleotide to the synthesis initiator.
  • synthesis of a polynucleotide comprises adding blocked nucleotides stepwise to an oligonucleotide bound to the reaction surface on the element via the cycled steps of: addition of nucleotide comprising a blocking group (i.e., a blocked nucleotide) to a synthesis initiator or extended polynucleotide comprising previously added nucleotides, binding of the nucleotide to the end of the synthesis initiator or extended polynucleotide catalyzed by the polymerase, and removal of the blocking group from the nucleotide to allow addition of a subsequent nucleotide to the extended polynucleotide.
  • a blocking group i.e., a blocked nucleotide
  • the blocking group bound to the nucleotide is a group capable of preventing addition of another nucleotide once the nucleotide has been added to the synthesis initiator or extended polynucleotide.
  • the extended polynucleotide is immersed in a nucleotide deblocking solution capable of removing the blocking group from the nucleotide.
  • the blocking group is the polymerase that catalyzes addition of the nucleotide to the surface-bound polynucleotide, wherein the polymerase is linked to the nucleotide (i.e., a nucleotide-polymerase conjugate).
  • the polymerase can sterically hinder addition of a subsequent nucleotide after addition of the blocked nucleotide to the polynucleotide.
  • a monomer deblocking solution that removes the polymerase from the nucleotide can then be used to remove the blocking group, such as a linker cleavage solution.
  • the blocking group is a reversible terminator bound to the nucleotide.
  • a monomer deblocking solution that removes the reversible terminator from the nucleotide can then be used to remove the blocking group, such as a linker cleavage solution.
  • both a reversible terminator and a polymerase bound to the nucleotide may be used.
  • Both the nucleotide addition and blocking group removal steps may be quenched by immersing the extended polynucleotide in an appropriate reaction quenching solution, such as EDTA. In addition, washing steps may be used between steps by immersing the extended polynucleotide in a wash buffer.
  • the present disclosure includes use of TdT with free nucleotides that have a 3′ modification to enable single extensions.
  • the present disclosure also includes use of TdT with a tethered nucleotide (we call this polymerase-nucleotide conjugate). Linkage of the dNTP can occur via a tether to the nucleobase.
  • a nucleotide comprises an optionally substituted O-alkyl group.
  • conjugates comprise the polymerase Terminal deoxynucleotidyl Transferase (TdT).
  • TdT Terminal deoxynucleotidyl Transferase
  • the method may employ conjugates comprising another template-independent polymerase.
  • FIG. 1A illustrates a typical process for the stepwise synthesis of a defined sequence using a template-independent polymerase.
  • a nucleic acid that serves as an initial substrate for elongation i.e., "starter molecule”
  • starter molecule a nucleic acid that serves as an initial substrate for elongation
  • first polymerase-nucleotide conjugate Once the nucleic acid has been elongated by the tethered nucleotide of a conjugate, no further elongations occur because the conjugates implement a termination mechanism.
  • the linker is cleaved to release the polymerase and reverse the termination mechanism, thus enabling subsequent elongations.
  • FIG. 1B illustrates a synthesis procedure using a conjugate comprising TdT and a photocleavable linker. As described above, other strategies are available for the attachment and cleavage of the linker.
  • tethered ribonucleoside triphosphates may be used.
  • an RNA specific nucleotidyl transferase such as E. coli Poly(A) Polymerase (IUBMB EC 2.7.7.19) or Poly(U) Polymerase, among others, may be employed.
  • RNA nucleotidyl transferases can contain modifications, e.g., single point mutations, that influence the substrate specificity towards a specific rNTP (Lunde et al., Nucleic acids research 40.19 (2012): 9815-9824.).
  • a very short tether between an RNA nucleotidyl transferase and a ribonucleoside triphosphate may be used to induce a high effective concentration of the nucleoside triphosphate, thereby forcing incorporation of an rNTP that might not be the natural substrate of the nucleotidyl transferase.
  • a conjugate comprising a polymerase and a nucleoside triphosphate
  • it preferentially elongates the nucleic acid using its tethered nucleotide (as opposed to using the nucleotide of another conjugate molecule).
  • the polymerase then remains attached to the nucleic acid via its tether to the added nucleotide until exposed to some stimulus that causes cleavage of the linkage to the added nucleotide.
  • the linker tethering the incorporated nucleotide to the polymerase can be cleaved, releasing the polymerase from the nucleic acid and therefore re-exposing its 3′ OH group for subsequent elongation.
  • Methods for nucleic acid synthesis provided herein that employ the shielding effect to achieve termination comprise an extension step wherein a nucleic acid is exposed to conjugates preferentially in the absence of free (i.e., untethered) nucleoside triphosphates, because the termination mechanism of shielding may not prevent their incorporation into the nucleic acid.
  • termination of further elongation may be "complete", meaning that after a nucleic acid molecule has been elongated by a conjugate, further elongations cannot occur during the reaction.
  • termination of further elongation may be "incomplete”, meaning that further elongations can occur during the reaction but at a substantially decreased rate compared to the initial elongation, e.g., 100 times slower, or 1000 times slower, or 10,000 times slower, or more.
  • Conjugates that achieve incomplete termination may still be used to extend a nucleic acid by predominantly a single nucleotide (e.g., in methods for nucleic acid synthesis and sequencing) when the reaction is stopped after an appropriate amount of time.
  • the reagent containing the conjugate may additionally contain polymerases without tethered nucleoside triphosphates, but those polymerases should not significantly affect the reaction because there are no free dNTPs in the mix.
  • Reagents based on conjugates employing the shielding effect to achieve termination preferentially only contain polymerase-nucleotide conjugates in which all polymerases remain folded in the active conformation. In some cases, if the polymerase moiety of a conjugate is unfolded, its tethered nucleoside triphosphate may become more accessible to the polymerase moieties of other conjugate molecules.
  • the unshielded nucleotides may be more readily incorporated by other conjugate molecules, circumventing the termination mechanism.
  • Polymerase-nucleotide conjugates employing the shielding effect to achieve termination are preferentially only labeled with a single nucleoside triphosphate moiety.
  • Polymerase nucleotide conjugates labeled with multiple nucleoside triphosphates that can access the catalytic site can, in some cases, incorporate multiple nucleoside triphosphates into the same nucleic acid. Additional tethered nucleotides may therefore lead to additional, undesired nucleotide incorporations into a nucleic acid during a reaction.
  • Polymerase-nucleotide conjugates employing the shielding effect to achieve termination preferentially comprise as short of a linker as possible that still enables the nucleoside triphosphate to frequently access the catalytic site of its tethered polymerase molecule in a productive conformation, in order to enable fast incorporation of the nucleotide into a nucleic acid.
  • Such conjugates may also preferentially employ an attachment position of the linker to the polymerase as close to the catalytic site as possible, enabling use of a shorter linker.
  • the length of the linker will determine the maximum distance from the attachment point a tethered nucleoside triphosphate or a tethered nucleic acid can reach. A smaller distance may lead to a reduced accessibility of the tethered moiety to other polymerase- nucleotide molecules, as discussed below.
  • linkers are approximately 24 and 28 ⁇ long.
  • Shorter linkers e.g., with lengths of 8-15 ⁇ may increase shielding; longer linkers, e.g., linkers longer than 50 ⁇ , 70 ⁇ or 100 ⁇ , may reduce shielding.
  • the shielding effect may be influenced by a combination of factors including, but not limited to, to the structure of the polymerase, the length of the linker, the structure of the linker, the attachment position of the linker to the polymerase, the binding affinity of the nucleoside triphosphate to the catalytic site of the polymerase, the binding affinity of the nucleic acid to the polymerase, the preferred conformation of the polymerase, and the preferred conformation of the linker.
  • One contribution to shielding can be steric effects that block the 3' OH of a nucleic acid that has been elongated by a conjugate from reaching into the catalytic site of another conjugate's polymerase moiety. Steric effects may also hinder a tethered nucleoside triphosphate from reaching into the catalytic site of another polymerase-nucleotide conjugate molecule due to clashes between the conjugates that would occur during such approaches.
  • steric effects may result in complete termination if they completely block productive interactions between the tethered nucleoside triphosphate (or elongated nucleic acid) of one conjugate molecule with another conjugate molecule, or may result in incomplete termination if they only hinder such intermolecular interactions.
  • Another contribution to shielding arises from the binding affinity of the tethered nucleoside triphosphate to the catalytic site of the polymerase
  • the tethered nucleoside triphosphate of a conjugate will have a high effective concentration with respect to the catalytic site of its tethered polymerase so it may remain bound to that site much of the time.
  • nucleoside triphosphate When the nucleoside triphosphate is bound to the catalytic site of its tethered polymerase molecule it is unavailable for incorporation by other polymerase molecules.
  • tethering reduces the effective concentration of nucleoside triphosphates available for intermolecular incorporation (i.e., incorporation catalyzed by a polymerase molecule to which the nucleotide is not tethered).
  • This shielding effect can enhance termination by reducing the rate by which a nucleic acid is elongated using the nucleoside triphosphate moiety of one conjugate molecule by the polymerase moiety of another conjugate molecule.
  • nucleic acid is tethered to the conjugate via its 3′ terminal nucleotide and will have a high effective concentration with respect to the catalytic site of its tethered polymerase so it may remain bound to that site much of the time.
  • nucleic acid is bound to the catalytic site of its tethered polymerase molecule it is unavailable for elongation by other conjugate molecules.
  • the polymerase-nucleotide conjugates comprise additional moieties that sterically hinder the tethered nucleoside triphosphate (or a tethered nucleic acid post-elongation) from approaching the catalytic sites of another conjugate molecule.
  • moieties include polypeptides or protein domains that can be inserted into a loop of the polymerase, and those and other bulky molecules such as polymers that can be site- specifically ligated e.g., to an inserted unnatural amino acid or specific polypeptide tag.
  • the linker is attached the 5 position of pyrimidines or the 7 position of 7-deazapurines.
  • the linker may be attached to an exocyclic amine of a nucleobase, e.g., by N-alkylating the exocyclic amine of cytosine with a nitrobenzyl moiety as discussed below.
  • the linker may be attached to any other atom in the nucleobase, sugar, or oc-phosphate, as will be apparent to those skilled in the art.
  • Certain polymerases have a high tolerance for modification of certain parts of a nucleotide, e.g., modifications of the 5 position of pyrimidines and the 7 position of purines are well-tolerated by some polymerases (He and Seela., Nucleic Acids Research 30.24 (2002): 5485-5496.; or Hottin et al., Chemistry. 2017 Feb 10;23(9):2109-2118).
  • the linker is attached to these positions.
  • a polymerase-nucleotide conjugate is prepared by first synthesizing an intermediate compound comprising a linker and a nucleoside triphosphate (referred to herein as a "linker-nucleotide"), and then this intermediate compound is attached to the polymerase.
  • nucleosides with substitutions compared to natural nucleosides e.g., pyrimidines with 5-hydroxymethyl or 5-propargylamino substituents, or 7- deazapurines with 7-hydroxymethyl or 7-propargylamino substituents may be useful starting materials for preparing linker- nucleotides.
  • nucleosides with 5- and 7- hydroxymethyl substituents that may be useful for preparing linker-nucleotides is shown below: [0276]
  • An exemplary set of nucleosides with 5- and 7-deaza-7-propargylamino substituents that may be useful for preparing linker-nucleotides is shown below: [0277]
  • These nucleosides are also commercially available as deoxyribonucleoside triphosphates.
  • the tethered nucleotide may be specifically attached to a cysteine residue of the polymerase using a sulfhydryl-specific attachment chemistry.
  • Possible sulfhydryl specific attachment chemistries include, but are not limited to ortho-pyridyl disulfide (OPSS), maleimide functionalities, 3- arylpropiolonitrile functionalities, allenamide functionalities, haloacetyl functionalities such as iodoacetyl or bromoacetyl, alkyl halides or perfluroaryl groups that can favorably react with sulfhydryls surrounded by a specific amino acid sequence (Zhang, Chi, et al. Nature chemistry 8, (2015) 120-128.).
  • OPSS ortho-pyridyl disulfide
  • maleimide functionalities 3- arylpropiolonitrile functionalities
  • allenamide functionalities such as iodoacetyl or bromoacetyl
  • alkyl halides or perfluroaryl groups that can favorably react with sulfhydryls surrounded by a specific amino acid sequence
  • the linker could be attached to a lysine residue via an amine - reactive functionality (e.g., NHS esters, Sulfo-NHS esters, tetra- or pentafluorophenyl esters, isothiocyanates, sulfonyl chlorides, etc.).
  • an amine - reactive functionality e.g., NHS esters, Sulfo-NHS esters, tetra- or pentafluorophenyl esters, isothiocyanates, sulfonyl chlorides, etc.
  • the linker may be attached to the polymerase via attachment to a genetically inserted unnatural amino acid, e.g., p- propargyloxyphenylalanine or p- azidophenylalanine that could undergo azide-alkyne Huisgen cycloaddition, though many suitable unnatural amino acids suitable for site-specific labeling exist and can be found in the literature (e.g., as described in Lang and Chin., Chemical reviews 114.9 (2014): 4764-4806.).
  • the linker may be specifically attached to the polymerase N- terminus.
  • the polymerase is mutated to have an N-terminal serine or threonine residue, which may be specifically oxidized to generate an N-terminal aldehyde for subsequent coupling to e.g., a hydrazide.
  • the polymerase is mutated to have an N-terminal cysteine residue that can be specifically labeled with an aldehyde to form a thiazolidine.
  • an N-terminal cysteine residue can be labeled with a peptide linker via Native Chemical Ligation.
  • a peptide tag sequence may be inserted into the polymerase that can be specifically labeled with a synthetic group by an enzyme, e.g., as demonstrated in the literature using biotin ligase, transglutaminase, lipoic acid ligase, bacterial sortase and phosphopantetheinyl transferase (e.g., as described in refs. 74-78 of Stephanopoulos & Francis Nat. Chem. Biol. 7, (2011) 876-884).
  • the linker is attached to a labeling domain fused to the polymerase.
  • a linker with a corresponding reactive moiety may be used to covalently label SNAP tags, CLIP tags, HaloTags and acyl carrier protein domains (e.g., as described in refs. 79-82 of Stephanopoulos & Francis Nat. Chem. Biol. 7, (2011) 876-884).
  • the linker is attached to an aldehyde specifically generated within the polymerase, as described in Carrico et al. (Nat. Chem. Biol. 3, (2007) 321 - 322).
  • FGE formylglycine-generating enzyme
  • FGE formylglycine-generating enzyme
  • This aldehyde may then be specifically labeled with e.g., a hydrazide or aminooxy moiety of a linker.
  • a linker may be attached to the polymerase via non-covalent binding of a moiety of the linker to a moiety fused to the polymerase.
  • attachment strategies include fusing a polymerase to streptavidin that can bind a biotin moiety of a linker, or fusing a polymerase to anti-digoxigenin that can bind a digoxigenin moiety of a linker.
  • site-specific labeling may lead to an attachment of the linker to the polymerase that may readily be reversed (e.g., an ortho-pyridyl disulfide (OPSS) group that forms a disulfide bond with a cysteine that can be cleaved using reducing agents, e.g., using TCEP), other attachment chemistries will produce permanent attachments.
  • OPSS ortho-pyridyl disulfide
  • the polymerase may be mutated to ensure specific attachment of the tethered nucleotide to a particular location of the polymerase, as will be apparent to those skilled in the art.
  • accessible cysteine residues in the wild-type polymerase may be mutated to a non-cysteine residue to prevent labeling at those positions.
  • a cysteine residue may be introduced by mutation at the desired attachment position. These mutations preferentially do not interfere with the activity of the polymerase.
  • a polymerase-nucleotide conjugate is prepared by first synthesizing an intermediate compound comprising a linker and a nucleotide (referred to herein as a "linker-nucleotide"), and then this intermediate compound is attached to the polymerase.
  • linker-nucleotide a linker-nucleotide
  • a person of ordinary skill in the art will understand the conjugates and nucleotides disclosed herein can be prepared in a manner similar to the reaction schemes shown below.
  • connection of the conjugate components can be achieved by the formation of a disulfide (forming a readily cleavable connection), formation of an amide, formation of an ester, protein-ligand linkage (e.g., biotin-streptavidin linkage), by alkylation (e.g., using a substituted iodoacetamide reagent) or forming adducts using aldehydes and amines or hydrazines [0290]
  • the separate components of the conjugate comprise a site suitable for conjugation to facilitate conjugate synthesis (i.e., a conjugate group).
  • conjugate groups include but are limited to hydroxyl, ester, amine, carbonate, acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, hydrazide, thiol, alkanoic acid, acid halide, isocyanate, isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal, dione, mesylate, tosylate, and tresylate.
  • conjugate groups include -NH2, -COOH, -COOCH3, -N- hydroxysuccinimide, and -maleimide.
  • the bioconjugate reactive group may be blocked (e.g., with a blocking group). Additional examples of bioconjugate reactive groups and the resulting bioconjugate reactive linkers can be found, e.g., in PCT Publication WO2021/226327, incorporated by reference in its entirety.
  • a desired nucleotide can be commercially obtained, and its hydroxyl groups protected by TBS before conjugation of the L1-OH group to an exocylic oxygen or amine on the nucleobase.
  • an already modified nucleotide comprising the L1-OH group bound to the nucleotide can be obtained, such as an L1-OH group bound to the C5 of a pyrimidine or an L1-OH group bound to C7 of a 7 deazapurine.
  • An Fmoc-protected amino acid ester is then coupled to the hydroxyl group of L1-OH, followed by hydroxyl group deprotection and triphosphorylation of the nucleoside.
  • linker Fmoc is removed from the amino acid ester amine group and it is coupled to the rest of the linker including L3, which is capable of binding to a polymerase.
  • the linker is bound to a portion of the nucleotide at an atom that is not involved in base pairing. In other embodiments, the linker is bound to the nucleobase of the nucleotide at an atom that is involved in base pairing.
  • the linker is considered to be at least the atoms that connect the polymerase to any atom in the monocyclic or polycyclic ring system bonded to the ⁇ position of the sugar (e.g., pyrimidine or purine or 7-deazapurine or 8-aza-7-deazapurine).
  • Certain polymerases have a high tolerance for modification of certain parts of a nucleotide, e.g., modifications of the 5 position of pyrimidines and the 7 position of purines are well-tolerated by some polymerases (He and Seela., Nucleic Acids Research 30.24 (2002): 54855496 ; or Hottin et al Chemistry 2017 Feb 10;23(9):2109 2118)
  • the linker is attached the 5 position of pyrimidines or the 7 position of 7- deazapurines.
  • the linker may be attached to an exocyclic amine of a nucleobase, e.g., by N-alkylating the exocyclic amine of cytosine with a nitrobenzyl moiety as discussed below.
  • the linker is joined to the sugar or to the ⁇ -phosphate of the nucleotide.
  • the linker is jointed to the terminal phosphate of the nucleotide.
  • the linker used should be sufficiently long to allow the nucleotide to access the active site of the polymerase to which it is tethered.
  • the polymerase of a conjugate is capable of catalyzing the addition of the nucleotide to which it is linked onto the 3′ end of a nucleic acid.
  • Conjugation of nucleotides or other base-pairing moieties to linkers may be achieved by any means known in the art of chemical conjugation methods. Nucleotide bases can be obtained or modified to include an L1 portion of the linker. The rest of the linker can be attached to L1 using methods exemplified herein. Those skilled in the art will know or be able to determine appropriate methods for attaching linkers based on the reactivities of these bases.
  • nucleotides containing base modifications that add a free amine group are contemplated for use in conjugation to linkers as described herein.
  • Primary amines for example, may be linked to the base in such a manner that they can be reacted with heterobifunctional polyethylene glycol (PEG) linkers to create a nucleotide containing a variable length PEG linker.
  • PEG polyethylene glycol
  • examples of such amine-containing nucleotides include 5- propargylamino-dNTPs, 5- propargylamino-NTPs, amino allyl-dNTPs, and amino allyl- NTPs.
  • the tethered nucleotide may be specifically attached to a cysteine residue of the polymerase using a sulfhydryl-specific attachment chemistry.
  • Possible sulfhydryl specific attachment chemistries include, but are not limited to ortho-pyridyl disulfide (OPSS), maleimide functionalities, 3- arylpropiolonitrile functionalities, allenamide functionalities, haloacetyl functionalities such as iodoacetyl or bromoacetyl, alkyl halides or perfluroaryl groups that can favorably react with sulfhydryls surrounded by a specific amino acid sequence (Zhang, Chi, et al.
  • OPSS ortho-pyridyl disulfide
  • maleimide functionalities 3- arylpropiolonitrile functionalities
  • allenamide functionalities haloacetyl functionalities
  • haloacetyl functionalities such as iodoacetyl or brom
  • the linker could be attached to a lysine residue via an amine - reactive functionality (e.g., NHS esters, Sulfo-NHS esters, tetra- or pentafluorophenyl esters, isothiocyanates, sulfonyl chlorides, etc.).
  • an amine - reactive functionality e.g., NHS esters, Sulfo-NHS esters, tetra- or pentafluorophenyl esters, isothiocyanates, sulfonyl chlorides, etc.
  • the linker may be attached to the polymerase via attachment to a genetically inserted unnatural amino acid, e.g., p- propargyloxyphenylalanine or p- azidophenylalanine that could undergo azide-alkyne Huisgen cycloaddition, though many suitable unnatural amino acids suitable for site-specific labeling exist and can be found in the literature (e.g., as described in Lang and Chin., Chemical reviews 114.9 (2014): 4764-4806.).
  • a genetically inserted unnatural amino acid e.g., p- propargyloxyphenylalanine or p- azidophenylalanine that could undergo azide-alkyne Huisgen cycloaddition
  • the linker may be specifically attached to the polymerase N- terminus.
  • the polymerase is mutated to have an N-terminal serine or threonine residue, which may be specifically oxidized to generate an N-terminal aldehyde for subsequent coupling to e.g., a hydrazide.
  • the polymerase is mutated to have an N-terminal cysteine residue that can be specifically labeled with an aldehyde to form a thiazolidine.
  • an N-terminal cysteine residue can be labeled with a peptide linker via Native Chemical Ligation.
  • a peptide tag sequence may be inserted into the polymerase that can be specifically labeled with a synthetic group by an enzyme, e.g., as demonstrated in the literature using biotin ligase, transglutaminase, lipoic acid ligase, bacterial sortase and phosphopantetheinyl transferase (e.g., as described in refs. 74-78 of Stephanopoulos & Francis Nat. Chem. Biol. 7, (2011) 876-884).
  • an enzyme e.g., as demonstrated in the literature using biotin ligase, transglutaminase, lipoic acid ligase, bacterial sortase and phosphopantetheinyl transferase (e.g., as described in refs. 74-78 of Stephanopoulos & Francis Nat. Chem. Biol. 7, (2011) 876-884).
  • the linker is attached to a labeling domain fused to the polymerase.
  • a linker with a corresponding reactive moiety may be used to covalently label SNAP tags, CLIP tags, HaloTags and acyl carrier protein domains (e.g., as described in refs. 79-82 of Stephanopoulos & Francis Nat. Chem. Biol. 7, (2011) 876-884).
  • the linker is attached to an aldehyde specifically generated within the polymerase, as described in Carrico et al. (Nat. Chem. Biol. 3, (2007) 321 - 322).
  • FGE formylglycine-generating enzyme
  • a linker may be attached to the polymerase via non-covalent binding of a moiety of the linker to a moiety fused to the polymerase.
  • attachment strategies include fusing a polymerase to streptavidin that can bind a biotin moiety of a linker, or fusing a polymerase to anti-digoxigenin that can bind a digoxigenin moiety of a linker.
  • site-specific labeling may lead to an attachment of the linker to the polymerase that may readily be reversed (e.g., an ortho-pyridyl disulfide (OPSS) group that forms a disulfide bond with a cysteine that can be cleaved using reducing agents, e.g., using TCEP), other attachment chemistries will produce permanent attachments.
  • OPSS ortho-pyridyl disulfide
  • the polymerase may be mutated to ensure specific attachment of the tethered nucleotide to a particular location of the polymerase, as will be apparent to those skilled in the art.
  • accessible cysteine residues in the wild-type polymerase may be mutated to a non-cysteine residue to prevent labeling at those positions.
  • a cysteine residue may be introduced by mutation at the desired attachment position. These mutations preferentially do not interfere with the activity of the polymerase.
  • the linker is specifically attached to an amino acid of the polymerase.
  • Residues known to be involved with catalysis and methods for determining if a residue is involved with catalysis will be apparent to those skilled in the art and are reviewed in literature (e.g.. Joyce et al. (Journal of Bacteriology 177.22 (1995): 6321.) and Jara and Martinez (The Journal of Physical Chemistry B 120.27 (2016): 6504-6514.))
  • the polymerase can be a template-independent polymerase, i.e., a terminal deoxynucleotidyl transferase or DNA nucleotidylexotransferase, which terms are used interchangeably to refer to an enzyme having activity 2.7.7.31 using the IUBMB nomenclature.
  • a description of such enzymes can be found in Bollum, F.J.
  • the polymerase is mutated to improve addition of the modified nucleotide.
  • any polymerase capable of extending a polynucleotide, incorporating a nucleotide into a polynucleotide, or incorporating a nucleotide analog into a polynucleotide is envisaged for use in the conjugates and methods described herein.
  • the polynucleotide is single stranded.
  • the polynucleotide is double stranded.
  • the polynucleotide is immobilized on a solid support.
  • DNA polymerases examples include polA, polB, polC, polD, polY, polX, reverse transcriptases (RT), and high-fidelity polymerases.
  • the polymerase is a modified polymerase.
  • the polymerase comprises 29, B103, GA-1, PZA, 15, BS32, M2Y, Nf, Gl, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, L17, ThermoSequenase®, 9°NmTM, TherminatorTM DNA polymerase, Tne, Tma, Tfl, Tth, TIi, Stoffel fragment, VentTM and Deep VentTM DNA polymerase, KOD DNA polymerase, Tgo, JDF-3, Pfu, Taq, T7 DNA polymerase, T7 RNA polymerase, PGB-D, UlTma DNA polymerase, E. coli DNA polymerase I, E.
  • coli DNA polymerase III archaeal DP1EDP2 DNA polymerase II, 9°N DNA Polymerase, Taq DNA polymerase, Phusion® DNA polymerase, Pfu DNA polymerase, SP6 RNA polymerase, RB69 DNA polymerase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, SuperScript® II reverse transcriptase, and SuperScript® III reverse transcriptase.
  • AMV Avian Myeloblastosis Virus
  • MMLV Moloney Murine Leukemia Virus
  • the polymerase is DNA polymerase 1 -KI enow fragment, Vent polymerase, Phusion® DNA polymerase, KOD DNA polymerase, Taq polymerase, T7 DNA polymerase, T7 RNA polymerase, TherminatorTM DNA polymerase, POLB polymerase, SP6 RNA polymerase, E. coli DNA polymerase I, E. coli DNA polymerase III, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, SuperScript® II reverse transcriptase, or SuperScript® III reverse transcriptase.
  • AMV Avian Myeloblastosis Virus
  • MMLV Moloney Murine Leukemia Virus
  • the polymerase molecules used in the methods described herein can be polymerase theta, a DNA polymerase, or any enzyme that can extend nucleotide chains.
  • the polymerase is tri29.
  • the polymerase is a protein with pockets that work around terminal phosphate groups, for example, a triphosphate group.
  • the described methods use TdT with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations to synthesize defined polynucleotides.
  • the described method uses TdT with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations to a surface-accessible amino acid residue.
  • the TdT is a variant of TdT.
  • the variant of TdT comprises a cysteine mutation.
  • the polymerase is mutated to improve addition of a modified nucleotide bound to the polymerase forming a conjugate.
  • the variant TdT comprises at least 70%, 80%, 90%, or 95% sequence identity to wild-type TdT.
  • the described methods use polymerase theta with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations to synthesize defined polynucleotides. In some embodiments, the described method uses polymerase theta with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations to a surface-accessible amino acid residue. In some embodiments, the polymerase theta is a variant of polymerase theta. In some instances, the variant polymerase theta comprises at least 70%, 80%, 90%, or 95% sequence identity to wild-type polymerase theta. In some embodiments, the polymerase theta is encoded by POLQ.
  • Enzymes described herein comprise one or more unnatural amino acids.
  • the unnatural amino acid comprises: a lysine analogue; an aromatic side chain; an azido group; an alkyne group; or an aldehyde or ketone group.
  • the unnatural amino acid does not comprise an aromatic side chain.
  • the unnatural amino acid is selected from N6-azidoethoxy-carbonyl- L-lysine (AzK), N6-propargylethoxy-carbonyl-L-lysine (PraK), N6-(propargyloxy)- carbonyl- L-lysine (PrK), p-azido-phenylalanine, BCN-L-lysine, norbornene lysine, TCO- lysine, methyltetrazine lysine, allyloxycarbonyllysine, 2-amino-8-oxononanoic acid, 2- amino-8- oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo- L-phenylalanine, m-acetylphenylalanine, 2-amino-8-ox
  • the polymerase is a fusion protein.
  • the fusion protein comprises maltose binding protein (MBP).
  • MBP maltose binding protein
  • TdT is fused to other enzymes such as helicase.
  • the polymerase comprises a template-independent polymerase.
  • the polymerase comprises a Pol-X family polymerase.
  • the polymerase comprises a Terminal deoxynucleotidyl Transferase (TdT), or a variant thereof.
  • the template-independent polymerase comprises a TdT or a variant thereof.
  • the TdT or variant thereof comprises a sequence sharing at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1.
  • the TdT comprises a sequence identical to SEQ ID NO: 1.
  • the TdT variant comprises one or more amino acid substitutions, insertions, or deletions to SEQ ID NO: 1.
  • TdT Terminal deoxynucleotidyl transferase: MGGRDIVDGSEFSPSPVPGSQNVPAPAVKKISQYACQRRTTLNNYNQLFTDALDILAENDEL RENEGSALAFMRASSVLKSLPFPITSMKDTEGIPSLGDKVKSIIEGIIEDGESSEAKAVLND ERYKSFKLFTSVFGVGLKTAEKWFRMGFRTLSKIQSDKSLRFTQMQKAGFLYYEDLVSCVNR PEAEAVSMLVKEAWTFLPDALVTMTGGFRRGKMTGHDVDFLITSPEATEDEEQQLLHKVTD FWKQQGLLLYADILESTFEKFKQPSRKVDALDHFQKCFLILKLDHGRVHSEKSGQQEGKGWK AIRVDLVMSPYDRRAFALLGWTGSRQFERDLRRYATHERKMMLDNHALYDRTKRVFLEAESE EEIFAHLGLDYIEPWERNA
  • template-independent polymerases having activity as described for E.C. class 2.7.7.31 are used.
  • the template-independent polymerase is a deoxynucleotidyl transferase or DNA nucleotidylexotransferase.
  • a description of such enzymes can be found in Bollum, F.J. Deoxynucleotide-polymerizing enzymes of calf thymus gland.
  • V Homogeneous terminal deoxynucleotidyl transferase. J. Biol. Chem. 246 (1971) 909-916; Gottesman, M.E. and Canellakis, E.S.
  • Additional polymerases with the ability to extend single stranded nucleic acids in the absence of template include, but are not limited to, Polymerase Theta (Kent et al., eLife 5 (2016): el3740.), polymerase mu (Juarez et al., Nucleic acids research 34.16 (2006): 4572-4582.; or McElhinny et all., Molecular cell 19.3 (2005): 357-366.) or polymerases where template independent activity is induced, e.g. by the insertion of elements of a template independent polymerase (Juarez et al., Nucleic acids research 34.16 (2006): 4572-4582).
  • the polymerase can be a template-dependent polymerase i.e., a DNA-directed DNA polymerase (e.g., an enzyme having activity 2.7.7.7 using the IUBMB nomenclature) or an RNA-directed DNA polymerase.
  • a DNA-directed DNA polymerase e.g., an enzyme having activity 2.7.7.7 using the IUBMB nomenclature
  • RNA-directed DNA polymerase e.g., an enzyme having activity 2.7.7.7 using the IUBMB nomenclature
  • the polymerase comprises an RNA polymerase.
  • an RNA specific nucleotidyl transferase such as E. coli Poly(A) Polymerase (IUBMB EC 2.7.7.19) or Poly(U) Polymerase, among others, may be employed.
  • the RNA nucleotidyl transferases can contain modifications, e.g., single point mutations, which influence the substrate specificity towards a specific rNTP (Lunde et al., Nucleic acids research 40.19 (2012): 9815-9824.).
  • a very short tether between an RNA nucleotidyl transferase and a ribonucleotide may be used to induce a high effective concentration of the nucleotide, thereby forcing incorporation of an rNTP that might not be the natural substrate of the nucleotidyl transferase.
  • a nucleotide is a ribose polyphosphate.
  • a ribose polyphosphate is selected from the group consisting of ribose triphosphate, ribose tetraphosphate, ribose pentaphosphate, and ribose hexaphosphate.
  • a ribose polyphosphate is a ribose triphosphate.
  • a ribose polyphosphate is a ribose hexaphosphate.
  • ribose polyphosphate is a ribose pentaphosphate.
  • ribose polyphosphate is a ribose tetraphosphate.
  • a nucleotide is a deoxyribose polyphosphate.
  • a deoxyribose polyphosphate is selected from the group consisting of deoxyribose triphosphate, deoxyribose tetraphosphate, deoxyribose pentaphosphate, and deoxyribose hexaphosphate.
  • a deoxyribose polyphosphate is a ribose triphosphate.
  • a deoxyribose polyphosphate is a deoxyribose hexaphosphate.
  • deoxyribose polyphosphate is a deoxyribose pentaphosphate.
  • deoxyribose polyphosphate is a deoxyribose tetraphosphate.
  • nucleotides refers to a molecule comprising an aromatic base, a five carbon sugar (e.g., ribose or deoxyribose), and at least one phosphate group.
  • a five carbon sugar e.g., ribose or deoxyribose
  • phosphate group e.g., ribose or deoxyribose
  • Canonical or non-canonical nucleotides are consistent with use of the term.
  • the phosphate in some embodiments comprises a monophosphate, diphosphate, or triphosphate, or corresponding phosphate analog.
  • Nucleotides typically comprise a hetero cyclic base including substituted or unsubstituted nitrogen-containing parent heteroaromatic ring which are commonly found in nucleic acids, including naturally-occurring, substituted, modified, or engineered variants, or analogs of the same.
  • Exemplary bases include, but are not limited to, purines and pyrimidines such as: 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N6-A2-isopentenyladenine (6iA), N6-A2-isopentenyl-2-methylthioadenine (2ms6iA), N6-methyladenine, guanine (G), isoguanine, N2-dimethylguanine (dmG), 7- methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and 06- methylguanine; 7-deaza-purines such as 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7- deaza-G); pyrimidines such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4- thiothymine
  • Nucleotides typically comprise a sugar moiety, such as carbocyclic moiety (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48), acyclic moieties (Martinez, et al., 1999 Nucleic Acids Research 27: 1271-1274; Martinez, et al., 1997 Bioorganic & Medicinal Chemistry Fetters vol. 7: 3013-3016), and other sugar moieties (Joeng, et al., 1993 J. Med. Chem. 36: 2627-2638; Kim, et al., 1993 J. Med. Chem. 36: 30-7; Eschenmosser 1999 Science 284:2118-2124; and U.S. Pat. No.
  • the sugar moiety comprises: ribosyl; 2'-deoxyribosyl; 3 '-deoxyribosyl; 2',3'-dideoxyribosyl; 2', 3'- didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'- fluororibosyl; 2'-mercaptoriboxyl; 2'-alkylthioribosyl; 3 '-alkoxyribosyl; 3 '-azidoribosyl; 3'- aminoribosyl; 3 '-fluororibosyl; 3'-mercaptoriboxyl; 3 '-alkylthioribosyl carbocyclic; acyclic or other modified sugars.
  • nucleotides comprise a chain of one, two or three phosphorus atoms where the chain is typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramide linkage.
  • the nucleotide is an analog having a phosphorus chain in which the phosphorus atoms are linked together with intervening O, S, NH, methylene or ethylene.
  • the phosphorus atoms in the chain include substituted side groups including O, S or BH3.
  • the chain includes phosphate groups substituted with analogs including phosphoramidate, phosphorothioate, phosphordithioate, and O-methylphosphoroamidite groups.
  • the polymerase of the conjugate may be covalently attached to oligonucleotides or nucleotides via the nucleotide base.
  • the nucleotide or oligonucleotide may have the polymerase attached to the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base through a linker moiety.
  • a nucleotide used in the present disclosure can also include native or non-native bases.
  • a native deoxyribonucleic acid can have one or more bases selected from the group consisting of adenine, thymine, cytosine or guanine and a ribonucleic acid can have one or more bases selected from the group consisting of uracil, adenine, cytosine or guanine.
  • Exemplary non-native bases that can be included in a nucleic acid, whether having a native backbone or analog structure include, without limitation, inosine, xathanine, hypoxathanine, isocytosine, isoguanine, 5- methylcytosine, 5-hydroxymethyl cytosine, 2-aminoadenine, 6- methyl adenine, 6-methyl guanine, 2-propyl guanine, 2-propyl adenine, 2-thioLiracil, 2- thiothymine, 2-thiocytosine, 15 - halouracil, 15 -halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil, 4-thiouracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8- thiol adenine or
  • the phosphorylated nucleoside (e.g., nucleotide) to be tethered to the polymerase is a nucleoside comprising at least one phosphate group.
  • the nucleoside comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or more than 9 phosphate groups.
  • the nucleoside comprises at least 3 phosphate groups.
  • the phosphorylated nucleoside is adenosine, cytidine, uridine, or guanosine, each of which comprises at least one phosphate group.
  • the phosphorylated nucleoside is a deoxy nucleoside comprising at least one phosphate group. In some embodiments, the phosphorylated nucleoside is a deoxynucleoside comprising at least 3 phosphate groups. In some embodiments, the deoxy nucleoside comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or more than 9 phosphate groups. In some embodiments, the phosphorylated nucleoside is deoxyadenosine, deoxycytidine, deoxythymidine, or deoxyguanosine, each of which comprises at least one phosphate group. In some embodiments, the phosphorylated nucleoside is a nucleoside triphosphate, such as dNTP.
  • the phosphorylated nucleoside is a nucleoside tetraphosphate, nucleoside pentaphosphate, a nucleoside hexaphosphate, a nucleoside heptaphosphate, nucleoside octaphosphate, or a nucleoside nonaphosphate.
  • the phosphorylated nucleoside is a nucleoside hexaphosphate.
  • the phosphorylated nucleoside is a nucleoside triphosphate.
  • the phosphorylated nucleoside is selected from the group consisting of deoxyadenosine triphosphate (dATP), deoxy guano sine triphosphate (dGTP), deoxy cytidine triphosphate (dCTP), deoxy thymidine triphosphate (dTTP), deoxyadenosine tetraphosphate, deoxyguanosine tetraphosphate, deoxycytidine tetraphosphate, deoxythymidine tetraphosphate, deoxyadenosine pentaphosphate, deoxyguanosine pentaphosphate, deoxycytidine pentaphosphate, deoxythymidine pentaphosphate, deoxyadenosine hexaphosphate, deoxyguanosine hexaphosphate, deoxycytidine hexaphosphate, deoxythymidine hexaphosphate, and any combination thereof.
  • dATP deoxyadenosine triphosphate
  • the nucleotides analogs described herein comprise a reversible terminator group, such as such as an O- azidomethyl or O-NH2 group on the 3' position of the sugar or an (alpha-tertbutyl-2- nitrobenzyl)oxymethyl group on the 5 position of pyrimidines or the 7 position of 7- deazapurines (for an overview see, e.g. Chen et al., Genomics, Proteomics & Bioinformatics 2013 11: 34-40).
  • the nucleotide analog prevents or hinders further elongation once incorporated into a nucleic acid to achieve controlled termination of synthesis.
  • the RTdNTP- polymerase conjugates when used as part of a conjugate, do not rely on the shielding effect to achieve termination, e.g.. when a 3' modified RTdNTP is tethered to the polymerase, the linker used may exceed 100 A or 200 A in length.
  • the linker is considered to be at least the atoms that connect the nucleotide to the polymerase.
  • the linker comprises atoms that connect the base, the sugar, or the oc-phosphate of a nucleotide to the polymerase.
  • the polymerase and the nucleotide are covalently linked and the distance between the linked atom of the nucleotide and the polymerase to which it is attached may be in the range of 4-100 A, e.g., 15-40 or 20-30 A, although this distance may vary depending on where the nucleoside triphosphate is tethered.
  • the linker may be a PEG or polypeptide linker, although, again, there is considerable flexibility on the type of linker used.
  • the linker should be joined to the base of the nucleotide at an atom that is not involved in base pairing.
  • the linker is considered to be at least the atoms that connect a Ca atom in the backbone of the polymerase to any atom in the monocyclic or polycyclic ring system bonded to the T position of the sugar (e.g.. pyrimidine or purine or 7-deazapurine or 8-aza-7-deazapurine).
  • the linker should be joined to the base of the nucleotide at an atom that is involved in base pairing.
  • the linker should be joined to the sugar or to the oc-phosphate of the nucleotide. In all embodiments, the linker used should be sufficiently long to allow the nucleoside triphosphate to access the active site of the polymerase to which it is tethered. As will be described in greater detail below, the polymerase of a conjugate is capable of catalyzing the addition of the nucleotide to which it is linked onto the 3' end of a nucleic acid.
  • the linker may be attached to various positions on the nucleotide, and a variety of cleavage strategies may be used. Those strategies may include, but are not limited to, the following examples:
  • the linker may be cleaved by exposure to a reducing agent such as dithiothreitol (DTT).
  • a linker comprising a 4-(disulfaneyl)butanoyloxy- methyl group attached to the 5 position of a pyrimidine or the 7 position of a 7-deazapurine may be cleaved by reducing agents (e.g.. DTT) to produce a 4-mercaptobutanoyloxymethyl scar on the nucleobase. This scar may undergo intramolecular thiolactonization to eliminate a 2-oxothiolane, leaving a smaller hydroxymethyl scar on the nucleobase.
  • reducing agents e.g.. DTT
  • An example of such a linker attached to the 5 position of cytosine is depicted below, but the strategy is applicable to any suitable nucleobase:
  • the linker may be cleaved by exposure to light.
  • a linker comprising (2-nitrobenzyl)oxymethyl group may be cleaved with 365 nm light, leaving a hydroxymethyl scar, e.g., as depicted for cytosine below, but as is applicable to any suitable nucleobase:
  • the linker may comprise a 3-(((2- nitrobenzyl)oxy)carbonyl)aminopropynyl group that may be cleaved with 365 nm light release a nucleobase with a propargylamino scar.
  • This strategy is applicable to any suitable nucleobase:
  • the linker may comprise an acyloxymethyl group that may be cleaved with a suitable esterase to release a nucleobase with a hydroxymethyl scar, e.g.. as depicted for cytosine below, but as is applicable to any suitable nucleobase:
  • the linker may comprise additional atoms (included in R' above) adjacent to the ester that increase the activity of the esterase towards the ester bond.
  • the linker may comprise an N-acyl-aminopropynyl group that may be cleaved with a peptidase to release a nucleobase with propargylamino scar, e.g., as depicted for 5 -propargylamino cytosine below, but as is applicable to any suitable nucleobase:
  • the linker may comprise additional atoms (included in R' above) adjacent to the amide that increase the activity of the peptidase towards the amide bond.
  • a polymerase-nucleotide conjugate comprises a nucleotide linked to a polymerase using an enzymatically cleavable linker.
  • a polymerase-nucleotide conjugate comprising an enzymatically cleavable linker comprises a structure Nuc-L 1 -L 2 -Pol, wherein Nuc represents a nucleotide, pol represents a polymerase, and L'-L 2 represents an enzymatically cleavable linker.
  • L 1 represents a region of an enzymatically cleavable linker connecting the nucleotide to L 2
  • L 2 represents a cleavable portion of an enzymatically cleavable linker.
  • L 2 also comprises a portion for connecting L 2 to Pol.
  • an enzymatically cleavable linker comprises an amino acid ester moiety.
  • L 2 comprises an amino acid ester moiety.
  • the ester group of an amino acid ester moiety is cleavable by a protease comprising esterase activity.
  • the ester of an amino acid of L 2 is attached to L 1 , which can also be referred to as a spacer or as a scar of a nucleotide after cleavage of the L 2 ester.
  • L 2 comprises attachment chemistry for polymerase conjugation.
  • L 2 further comprises additional amino acids bound to the amine of the amino acid ester to serve as a protease substrate.
  • L 2 is optimized for ester stability to prevent spontaneous cleavage while retaining the ability to act as a suitable substrate for esterase activity of a protease comprising esterase activity.
  • the amino acid ester comprises one or more substitutions at the alpha carbon, such as addition of an aliphatic or bulky substituent.
  • the amino acid ester is represented by: wherein R 1 and R 1 are each independently selected from an optionally substituted C1-3 alkyl, a halogen, or are optionally taken together with the atom on which they are attached to form an optionally substituted C3-C7 carbocyclic ring.
  • L 2 linker structures with different substituents on the alpha carbon of the amino acid ester are shown below (with L 3 representing a portion of the L 2 linker including attached to the polymerase):
  • L 2 comprises an amino acid ester adjacent to one or more amino acid residues.
  • the one or more amino acid residues are bound to the amine group of the amino acid ester.
  • L 2 comprises or consists of: wherein R 1 and R 1' are independently selected from hydrogen or an optionally substituted C1-3 alkyl or are taken together with the atom on which they are attached to form an optionally substituted C 3 -C 7 carbocyclic ring; each R 3 is an optionally substituted group independently selected from hydrogen, C1-6 alkyl, benzyl, -OH, -O(C1-6 alkyl), and -CN; each R c is hydrogen or optionally substituted C 1-6 alkyl; and n is 1, 2, or 3.
  • the one or more amino acids linked to the amine of the amino acid ester comprise L- or D- isomers of amino acid residues.
  • Naturally-occurring amino acid refers to Ala, Asp, Cys, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, Tyr, or citrulline.
  • D- designates an amino acid having the “D” (dextrorotary) configuration, as opposed to the configuration in the naturally occurring (“L-”) amino acids.
  • the amino acids described herein can be purchased commercially (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art.
  • amino acids with non-natural or artificial side chains are linked to the amine of the amino acid ester.
  • the one or more amino acids included in the L 2 portion of the linker / bound to the amino acid ester can be selected, for example, to optimize protease binding and ester cleavage.
  • a combinatorial library can be generated to test optimal cleavage activity, amino acids can be chosen based on existing known peptide sequence targets for the protease.
  • the protease comprising esterase activity can recognize the peptide portion of the linker and hydrolyzes the ester group of the amino acid ester of L 2 , resulting in removal of polymerase attached to the nucleotide via the linker, as disclosed herein.
  • a spacer can be used between the nucleotide and the linker, or between the linker and the label.
  • spacers can be used in order to increase L 2 availability towards the protease/esterase and increase the efficiency and fidelity of polymerases.
  • Exemplary spacers include, for example, polyethyleneglycol or other suitable spacers.
  • linkers comprising L 2 structures including an amino acid ester bound to one or more amino acid residues is shown below, with ‘L 3 ’ representing a portion of the L 2 cleavable linker that is capable of binding to the polymerase:
  • linker structures may include, but are not limited to, carbon-chain linkers (e.g., C6, C12, C18, C24, etc.), peptide linkers (e.g., poly-glycine or poly-alanine ranging from about 1 residue to about 1,000 residues in length), or polyether linkers (e.g., PEG, PPG, PAG, PTMG from about 1 polyether unit to about 1,000 polyether units in length).
  • carbon-chain linkers e.g., C6, C12, C18, C24, etc.
  • peptide linkers e.g., poly-glycine or poly-alanine ranging from about 1 residue to about 1,000 residues in length
  • polyether linkers e.g., PEG, PPG, PAG, PTMG from about 1 polyether unit to about 1,000 polyether units in length.
  • the linker comprises a chain of atoms selected from C, N, O, S, Si, and P, preferably having 0-500 atoms, wherein L 1 covalently connects to Nuc and L 2 , and wherein L 2 is covalently attached to Pol.
  • the atoms used in forming L 1 or including in L 2 may be combined in all chemically relevant ways, such as forming alkylene, alkenylene, and alkynylene, carbamates, carbonates, ethers, polyoxyalkylene, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids, peptides, acyloxylamines, hydroxamic acids, or combination above thereof.
  • the linker comprises one or more carbon atoms, zero, one, or more oxygen atoms, zero, one or more nitrogen atoms, zero, one, or more sulfur atoms, or a combination thereof, in different embodiments.
  • the linker comprises, comprises about, comprises at least, comprises at least about, comprises at most, or comprises at most about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3
  • the linker comprises a polymer, such as a homopolymer or a heteropolymer.
  • the linker comprise a plurality of repeat units.
  • the plurality of repeating units comprises identical repeating units.
  • the plurality of repeating units comprises two or more different repeating units.
  • the plurality of repeating units can comprise a polyether such as paraformaldehyde, polyethylene glycol (PEG), polypropylene glycol (PPG), polyalkylene glycol (PAG), polytetramethylene glycol (PTMG), or a combination thereof.
  • the plurality of repeating units can comprise PEGix, PEG23, PEG24, or a combination thereof.
  • the plurality of repeating units can comprise a polyalkylene, such as polyethene, polypropene, polybutene, or a combination thereof.
  • a repeating unit of the plurality of repeating units comprises no aromatic group.
  • a repeating unit of the plurality of repeating units comprises one or more aromatic groups.
  • the linker comprises any number of basic chemical starting blocks.
  • linkers may comprise linear or branched alkyl, alkenyl, or alkynyl chains, or combinations thereof, that provide a useful distance between the nucleotide and polymerase, the nucleotide and L 2 , or the polymerase and the cleavable moiety of L 2 .
  • amino-alkyl linkers e.g., amino-hexyl linkers
  • the longest chain of such linkers may include as many as 2 atoms, 3 atoms, 4 atoms, 5 atoms, 6 atoms, 7 atoms, 8 atoms, 9 atoms, 10 atoms, or even 11-35 atoms, or even 35-50 atoms.
  • the linear or branched linker may also contain heteroatoms other than carbon, including, but not limited to, oxygen, sulfur, phosphate, and nitrogen.
  • a polyoxyethylene chain (also commonly referred to as polyethyleneglycol, or PEG) is a preferred linker constituent due to the hydrophilic properties associated with polyoxyethylene. Insertion of heteroatom such as nitrogen and oxygen into the linkers may affect the solubility and stability of the linkers.
  • the linker may be rigid in nature or flexible. Rigid structures include laterally rigid chemical groups, e.g., ring structures such as aromatic compounds, multiple chemical bonds between adjacent groups, e.g., double or triple bonds, in order to prevent rotation of groups relative to each other, and the consequent flexibility that imparts to the overall linker. Thus, the degree of desired rigidity may be modified depending on the content of the linker, or the number of bonds between the individual atoms comprising the linker.
  • Ringed structures may include aromatic or non-aromatic rings. Rings may be anywhere from 3 carbons, to 4 carbons, to 5 carbons or even 6 carbons in size. Rings may also optionally include heteroatoms such as oxygen or nitrogen and also be aromatic or non-aromatic. Rings may additionally optionally be substituted by other alkyl groups and/or substituted alkyl groups.
  • Linkers that comprise ring or aromatic structures can include, for example aryl alkynes and aryl amides. Other examples of the linkers of the disclosure include oligopeptide linkers that also may optionally include ring structures within their structure.
  • the linker comprises is a C1-C10 alkylene chain, wherein 1-6 methylene units are optionally and independently replaced by -NH-, -O-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, -SS-, optionally substituted cycloalkylene (e.g., C3-C8, C3-C6, or C5-C6), optionally substituted heterocycloalkylene (e.g., 3 to 8, 3 to 6, or 5 to 6 membered), optionally substituted arylene (e.g., C6-C10, C10, or phenylene), or substituted or unsubstituted heteroarylene (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).
  • cycloalkylene e.g., C3-C8, C3-C6, or C5-C6
  • L 1 is a bond, -NH-, -O-, -C(O)-, -C(O)NH-, -NHC(O)-, - NHC(O)NH-, -C(O)O-, -OC(O)-, -SS-, optionally substituted alkylene (e.g., C1- C20, C10- C20, C1-C8, C1-C6, or C1-C4), optionally substituted heteroalkylene (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), optionally substituted cycloalkylene (e.g., C 3 -C 8 , C 3 - C6, or C5-C6), optionally substituted heterocycloalkylene (e.g., 3 to 8, 3 to 6, or 5 to 6 membered), optionally substituted (e.g., C6-C10, C10, or phenylene),
  • alkylene e.
  • L 1 is optionally substitutedC1-C20 alkylene. In some embodiments, L 1 is optionally substituted 2 to 20 membered heteroalkylene. In some embodiments, L 1 is optionally substituted C3-C8 cycloalkylene. In some embodiments, L 1 is optionally substituted 3 to 8 membered heterocycloalkylene. In embodiments, L 1 is optionally substituted C 6 -C 10 arylene. In embodiments, L 1 is optionally substituted 5 to 10 membered heteroarylene. [0363] In some embodiments, L 1 is substituted with 1-6 instances of R L .
  • Each R L is independently selected from the group consisting of oxo, halogen, -CCI 3 , -CBr 3 , -CF 3 , -CI 3 , - CN, -OH, -NH 2 , -COOH, -CONH 2 , -NO 2 , -SH, -SO 3 H, -SO 4 H, -SO 2 NH 2 , -NHNH 2 , -ONH 2 , - NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, - OCF3, -OCBr3, -OCI2, -OCHCI2, -OCHBr2, -OCHb, -OCHF2, -N3, optionally substituted alkyl (e.g., C 1 -C 20 , C 10 -C 20 , C 1 -C 8
  • L1 acts as an attachment point to the nucleotide and includes a hydroxyl terminal group which binds to a portion of L2 during synthesis.
  • L1 is a scar that is enzymatically cleavable after cleavage of polymerase-nucleotide linker / removal of the L2-pol moiety.
  • L2 comprises a bioconjugate group suitable for conjugation of L2 to the polymerase.
  • the bioconjugate group is an N-hydroxysuccinimide ester (NHS) group.
  • the bioconjugate group is a maleimide group.
  • the linker may then be covalently attached to the polymerase by reaction of the maleimide group with a cysteine residue of the polymerase.
  • the polymerase may be operably linked to a linker moiety including a covalent or non-covalent bond; amino acid tag (e.g., poly-amino acid tag, poly- His tag, 6His-tag (SEQ ID NO: 53)); chemical compound (e.g., polyethylene glycol); protein-protein binding pair (e.g., biotin-avidin); affinity coupling; capture probes; or any combination of these.
  • the linker moiety can be separate from or part of a polymerase variant.
  • nucleic acid synthesis can refer to synthesis, or generation of a product that is a nucleic acid molecule (e.g., a polynucleotide).
  • Methods of nucleic acid synthesis can comprise stepwise synthesis, wherein nucleotides are inserted stepwise into a nucleic acid polymer or polynucleotide.
  • one typical process for stepwise synthesis of a polynucleotide comprises adding nucleotides stepwise to a starter molecule (e.g., an initial oligonucleotide) via the cycled steps of: addition of a polymerase-nucleotide conjugate to an oligonucleotide under conditions suitable for covalently binding the nucleotide to the end of the oligonucleotide catalyzed by the polymerase.
  • a starter molecule e.g., an initial oligonucleotide
  • this method comprises: incubating a nucleic acid with a first conjugate under conditions in which the polymerase catalyzes the covalent addition of the nucleotide of the first conjugate onto the 3' hydroxyl of the nucleic acid, to make an extension product.
  • This reaction can be performed using a nucleic acid that is attached to a solid support or that is in solution, e.g., not tethered to a solid support.
  • the method comprises a deblocking (de-shielded) step wherein the cleavable linkage of the linker is cleaved, thereby releasing the polymerase from the extension product. Cleavage of the linker removes the polymerase to produce a deblocked extension product. Deblocking enables subsequent extension of the nucleic acid, and thus allows these steps to be repeated cyclically to produce an extension product of defined sequence.
  • the method may further comprise, after deblocking: incubating the deblocked extension product with a second conjugate under conditions in which polymerase catalyzes the covalent addition of the nucleotide of the second conjugate onto the 3' end of the extension product.
  • the method may involve (a) incubating a nucleic acid with a first conjugate under conditions in which the polymerase catalyzes the covalent addition of the nucleotide of the first conjugate (i.e., a single nucleotide) onto the 3' hydroxyl of the nucleic acid, to make an extension product; (b) cleaving the cleavable linkage of the linker, thereby releasing the polymerase from the extension product and deblocking the extension product; (c) incubating the deblocked extension product with a second conjugate of claim 1 under conditions in which the polymerase catalyzes the covalent addition of the nucleotide of the second conjugate onto the 3' end of the extension product, to make a second extension product; (d) repeating steps (b)-(c) on the second extension product multiple times (e.g., 2 to 100 or more times) to produce an extended oligonucleotide of a defined sequence.
  • the polymerase cataly
  • Steps (b) - (c) may be repeated as many times as necessary until an extension product of a defined sequence and length is synthesized.
  • the end product may be 2-100 bases in length, although, in theory, the method can be used to produce products of any length, including greater than 200 bases or greater than 500 bases.
  • methods of nucleic acid synthesis as provided herein are carried out in a reaction buffer composition.
  • the reaction buffer composition is an aqueous solution.
  • the reaction buffer composition comprises a set of components suitable for the stability of the polymerase, nucleotide, polymerase-nucleotide conjugates, starter molecule, nucleic acid molecule products, and any surface or matrix on which the methods disclosed herein are carried out.
  • the reaction buffer composition comprises a set of components suitable for carrying out catalytic steps (e.g., polynucleotide polymerization performed by a polymerase) described in methods of nucleic acid synthesis in accordance with the present disclosure.
  • catalytic steps e.g., polynucleotide polymerization performed by a polymerase
  • the conditions under which nucleic acid synthesis is carried out can be varied.
  • methods of nucleic acid synthesis in accordance with the present disclosure generate a nucleic acid molecule product (i.e., a polynucleotide product).
  • the nucleic molecule product i.e., polynucleotide product
  • a “target” or “pre-determined” sequence refers to a desired polynucleotide sequence that is intentionally produced by the method of nucleic acid synthesis.
  • the pre-determined sequence can include any number of nucleotides comprising a nucleobase (e.g., adenine, thymine, guanine, cytosine, and/or uracil).
  • the nucleotide is a modified nucleotide (i.e., a nucleotide analog).
  • the nucleobase is a modified nucleobase.
  • the pre-determined sequence contains one or more designated positions which may be a random nucleobase. Inclusion of a position with a random nucleobase can be useful, for example, in introducing randomized mutation into a polynucleotide product.
  • the present disclosure includes a method of synthesizing a polynucleotide comprising contacting a precursor polynucleotide with a conjugate comprising a nucleotide covalently linked to a polymerase via a cleavable linker, wherein said nucleotide comprises said blocked nucleobase.
  • the method of synthesizing a polynucleotide comprises cleaving a cleavable linker after addition of a nucleotide to a precursor polynucleotide.
  • the method of synthesizing a polynucleotide comprises repeating contacting, adding, and optionally cleaving steps described herein one or more times.
  • removal of one or more blocking groups described herein comprises contacting said polynucleotide with an enzyme capable of removing said one or more blocking groups from said blocked nucleobases.
  • a method of synthesizing a polypeptide comprising contacting said polynucleotide with two or more enzymes capable of removing said one or more blocking groups from said blocked nucleobases.
  • synthesis of a polynucleotide comprises adding nucleotides stepwise to a starter molecule (e.g., an initial oligonucleotide) via the cycled steps of: addition of polymerase-nucleotide conjugate to an oligonucleotide, binding of the nucleotide to the 3’ end of the oligonucleotide catalyzed by the polymerase, and cleavage of the polymerase from the added nucleotide. These steps can be repeated until a desired polynucleotide is synthesized.
  • a starter molecule e.g., an initial oligonucleotide
  • nucleotides comprising protected nucleobases helps to improve the efficiency and accuracy of synthesis by inhibiting secondary structure formation which can interfere with the addition of the incoming nucleotide by the polymerase during synthesis.
  • synthesis can be completed entirely with protected nucleotides, synthesis with a combination of unmodified and protected nucleotides can also be used effectively to improve polynucleotide synthesis.
  • only one of the four nucleotides added e.g., from G or T
  • protected nucleotides are only added at targeted positions where secondary structure or ternary structure is predicted, which could interfere with synthesis.
  • synthesis of a completed polynucleotide where synthesis is improved can include the use of only 1 or 2 protected nucleotides.
  • about 5%, about 10%, about 20%, about 30%, about 50%, substantially all, or 100% of a specific nucleotide is incorporated into the polynucleotide in their protected version.
  • less than 5%, less than 10%, less than 20%, less than 30%, or less than 50% of a specific nucleotide is incorporated into the polynucleotide in its protected version.
  • nucleic acid molecule product or polynucleotide product generated by the methods described herein can contain a plurality of products.
  • the plurality of products comprises a nucleic acid molecule comprising the target (i.e., pre-determined) sequence. In some embodiments, the plurality of products comprises a nucleic acid molecule comprising a sequence that is not the target sequence. In some embodiments, the plurality of products comprises a nucleic acid molecule product comprising the target sequence and a nucleic acid molecule product that is not the target sequence.
  • the “purity” of the plurality of products can refer to the ratio of the abundance of nucleic acid molecule products with the target sequence to the abundance of nucleic acid molecule products that do not have the target sequence. The purity of a product can be assessed by any number of methods known in the art for determining the sequence of a nucleic acid.
  • a method of nucleic acid synthesis in accordance with the present disclosure produces a product having a purity between about 10% and about 99.99%.
  • the method of nucleic acid synthesis produces a product having a purity of at least 10%.
  • the method of nucleic acid synthesis produces a product having a purity of at least 10%.
  • the method of nucleic acid synthesis produces a product having a purity of at least 20%. In some embodiments, the method of nucleic acid synthesis produces a product having a purity of at least 30%. In some embodiments, the method of nucleic acid synthesis produces a product having a purity of at least 40%. In some embodiments, the method of nucleic acid synthesis produces a product having a purity of at least 50%. In some embodiments, the method of nucleic acid synthesis produces a product having a purity of at least 60%. In some embodiments, the method of nucleic acid synthesis produces a product having a purity of at least 70%. In some embodiments, the method of nucleic acid synthesis produces a product having a purity of at least 80%.
  • the method of nucleic acid synthesis produces a product having a purity of at least 90%. In some embodiments, the method of nucleic acid synthesis produces a product having a purity of at least 95%. In some embodiments, the method of nucleic acid synthesis produces a product having a purity of at least 99%.
  • the nucleoside triphosphate may be a deoxyribonucleoside triphosphate or a ribonucleoside triphosphate.
  • a conjugate may comprise an RNA polymerase linked to a ribonucleoside triphosphate.
  • the nucleotide added to the nucleic acid may be a ribonucleotide.
  • a conjugate comprises an DNA polymerase linked to a deoxyribonucleoside triphosphate.
  • the nucleotide added to the nucleic acid may be a deoxyribonucleotide.
  • the nucleotide is a nucleotide analog.
  • the nucleotide analog is a reversible terminator. Reversible terminators are known in the art for use in nucleic acid synthesis.
  • the nucleotide may comprise a reversible terminator (“RTdNTP”) and the deblocking step of the method further comprises removing the blocking group (e.g., removing the terminator group) from the added nucleotide to produce the deblocked extension product.
  • RdNTP reversible terminator
  • Deblocking enables subsequent extension of the nucleic acid, and thus allows these steps to be repeated cyclically to produce an extension product of defined sequence.
  • These methods may comprise incubating a duplex comprising a primer and a template with a composition comprising a set of conjugates, wherein the conjugates correspond to G, A, T and C and are distinguishably labeled, e.g., fluorescently labeled; detecting which nucleotide has been added to the primer by detecting a label that is tethered to the polymerase that has added the nucleotide to the primer; deblocking the extension product by cleaving the linker; and repeating the incubation, detection and deblocking steps to obtain the sequence of at least part of the template.
  • a composition comprising a set of conjugates, wherein the conjugates correspond to G, A, T and C and are distinguishably labeled, e.g., fluorescently labeled
  • the present disclosure describes a method of enzymatic polynucleotide synthesis using polymerase-nucleotide conjugates to control the iterative addition of a single nucleotide per cycle onto the 3′ hydroxyl terminus of a growing polynucleotide strand via the nucleotide-bound polymerase to perform polynucleotide synthesis.
  • control is achieved through a so-called “shielding effect”.
  • Shielding describes the steric hinderance that prevents the 3′ hydroxyl terminus that has been elongated by a conjugate from being accessed by another conjugate while the polymerase remains attached to the added nucleotide, as well as the preventing the polymerase tethered to the nucleotide at the 3′ terminus from accessing the nucleotides of other conjugates.
  • a nucleic acid that serves as an initial substrate for elongation (i.e., "starter molecule") is incubated with a first polymerase-nucleotide conjugate.
  • starter molecule a nucleic acid that serves as an initial substrate for elongation
  • the linker is cleaved to release the polymerase and reverse the termination mechanism, thus enabling subsequent elongations.
  • the elongation products are then exposed to the second conjugate, and these two steps are iterated to elongate the nucleic acid by a defined sequence.
  • WO2017/223517 Also described in WO2017/223517 is a synthesis procedure using a conjugate comprising TdT and a photocleavable linker. As described above, other strategies are available for the attachment and cleavage of the linker.
  • An important step in this approach to polynucleotide synthesis is deblocking, or the removal of the tethered polymerase from the extended polynucleotide, making the 3′ terminus available for continued extension in the next cycle of synthesis.
  • the removal of the tethered polymerase preferably occurs with rapid kinetics to reduce synthesis cycle time, while also being performed under benign conditions to prevent damage to the polynucleotide being synthesized.
  • the removal of the tethered polymerase is also preferred to proceed to full completion, and to produce a cleavage product which does not impede continued extension or downstream applications of the complete DNA synthesis product.
  • the tether also allows for efficient conjugation of the nucleotide to the polymerase, and subsequently positions the nucleotide effectively within the active site to promote rapid incorporation to a free primer 3′ terminus.
  • cleavable linker designs used for the tethering of polymerases to nucleotides that are highly stable during storage and under oligo synthesis reaction conditions (before controlled linker cleavage), and enzymatically cleavable to completion in a short timeframe suitable for oligo synthesis.
  • a conjugate comprising a polymerase and a nucleotide linked via a linker that comprises an enzymatically cleavable linkage.
  • the polymerase moiety of a conjugate can elongate a nucleic acid using its linked nucleotide (i.e., the polymerase can catalyze the attachment of a nucleotide to which it is joined onto a nucleic acid) and remains attached to the elongated nucleic acid via the linker until the linker is enzymatically cleaved.
  • the linker comprises the atoms that connect the nucleotide to the polymerase.
  • the linker connects the base, the sugar, or the ⁇ - phosphate of a nucleotide to the polymerase.
  • the linker connects the terminal phosphate of a nucleotide to the polymerase. In some embodiments, the linker connects the nucleotide to the C ⁇ atom in the backbone of the polymerase. In some embodiments, the polymerase and the nucleotide are covalently linked and the distance between the linked atom of the nucleotide and the polymerase to which it is attached may be in the range of 4-100 ⁇ , e.g., 15-40 ⁇ or 20-30 ⁇ , although this distance may vary depending on where the nucleotide is tethered. The linker used should be sufficiently long to allow the nucleotide to access the active site of the polymerase to which it is tethered.
  • the polymerase of a conjugate is capable of catalyzing the addition of the nucleotide to which it is linked onto the 3′ end of a nucleic acid.
  • Linkers contemplated herein are also of sufficient length and stability to allow efficient hydrolysis by enzymatic means.
  • the number of carbons or atom in a linker, optionally derivatized by other functional groups, must be of sufficient length to allow either enzymatic cleavage of the polymerase from the nucleotide.
  • a cleavable linker comprises an amino acid ester.
  • an amino acid ester is the site of cleavage of the linker, thereby facilitating release of a polymerase upon exposure to an esterase or protease comprising esterase activity.
  • a portion of the cleavable linker comprising the amino acid ester is referred to herein as the “L 2 ” portion of the linker.
  • L 2 can be designed and optimized for enzymatic cleavage by an esterase or protease comprising esterase activity, for example, by modifying the chemical group attached to the alpha carbon of the amino acid ester, or by including one or more amino acids adjacent to the amino acid ester as part of L 2 .
  • a polymerase-nucleotide conjugate comprising cleavable linkers that are highly stable and rapidly enzymatically cleavable by proteases comprising esterase activity.
  • a polymerase-nucleotide conjugate comprises a nucleotide linked to a polymerase using an enzymatically cleavable linker.
  • a polymerase-nucleotide conjugate comprising an enzymatically cleavable linker comprises a structure Nuc-L 1 -L 2 -L 3 -Pol, wherein Nuc represents a nucleotide, pol represents a polymerase, and L 1 -L 2 -L 3 represents an enzymatically cleavable linker.
  • L 1 represents a region of an enzymatically cleavable linker connecting the nucleotide to L 2
  • L 2 represents a cleavable portion of an enzymatically cleavable linker
  • L 3 represents a region of an enzymatically cleavable linker connecting L 2 to Pol.
  • an enzymatically cleavable linker comprises an amino acid ester moiety.
  • L 2 comprises an amino acid ester moiety.
  • the ester group of an amino acid ester moiety is cleavable by a protease comprising esterase activity.
  • the ester of an amino acid of L 2 is attached to L 1 , which can also be referred to as a spacer or as a scar of a nucleotide after cleavage of the L 2 ester.
  • L 2 is also attached to L 3 , the rest of the linker, which comprises attachment chemistry for polymerase conjugation.
  • L 3 can also include or be referred to as a spacer.
  • L 2 further comprises additional amino acids bound to the amine of the amino acid ester to serve as a protease substrate.
  • L 2 is optimized for ester stability to prevent spontaneous cleavage while retaining the ability to act as a suitable substrate for esterase activity of a protease comprising esterase activity.
  • the linker is bound to the nucleotide at the nucleobase. In some embodiments, the linker is bound to the nucleotide at the sugar. In some embodiments, the linker is bound to the nucleotide at a 5′ phosphate group, wherein the nucleotide is any nucleoside polyphosphate. In some embodiments, the linker is bound to the alpha phosphate. In some embodiments, the linker is bound to the gamma, beta, delta, epsilon, zeta, eta, or theta phosphate. In some embodiments, the linker is bound to the terminal phosphate.
  • a linker of a conjugate may be attached to the 7-position of deaza dGTP or the 5-position of dTTP or dUTP.
  • Additional tethered nucleotides can be found, e.g., in PCT Publication WO2017/223517 “Nucleic Acid Synthesis and Sequencing Using Tethered Nucleoside Triphosphates,” the entirety of which is incorporated by reference.
  • the tethered nucleotide may be specifically attached to a cysteine residue of the polymerase using a sulfhydryl-specific attachment chemistry.
  • Possible sulfhydryl specific attachment chemistries include, but are not limited to ortho-pyridyl disulfide (OPSS), maleimide functionalities, 3- arylpropiolonitrile functionalities, allenamide functionalities, haloacetyl functionalities such as iodoacetyl or bromoacetyl, alkyl halides or perfluroaryl groups that can favorably react with sulfhydryls surrounded by a specific amino acid sequence (Zhang, Chi, et al. Nature chemistry 8, (2015) 120-128.).
  • OPSS ortho-pyridyl disulfide
  • maleimide functionalities 3- arylpropiolonitrile functionalities
  • allenamide functionalities such as iodoacetyl or bromoacetyl
  • alkyl halides or perfluroaryl groups that can favorably react with sulfhydryls surrounded by a specific amino acid sequence
  • L 2 comprises an amino acid ester.
  • the amino acid ester is the site of cleavage of the linker, facilitating the release of the polymerase from the nucleotide.
  • the ester group of a glycine amino acid ester in the linker could be unstable, resulting in spontaneous cleavage of the conjugate and unwanted nucleotide insertions during conjugate-based oligonucleotide synthesis (see Examples 2 and 3).
  • the amino acid ester comprises one or more substitutions at the alpha carbon, such as addition of an aliphatic or bulky substituent.
  • the amino acid ester is represented by: wherein R 1 and R 1' are each independently selected from an optionally substituted C1-3 alkyl, a halogen, or are optionally taken together with the atom on which they are attached to form an optionally substituted C 3 -C 7 carbocyclic ring.
  • R 1 and R 1' are each independently selected from an optionally substituted C1-3 alkyl, a halogen, or are optionally taken together with the atom on which they are attached to form an optionally substituted C 3 -C 7 carbocyclic ring.
  • L 2 comprises an amino acid ester adjacent to one or more amino acid residues.
  • the one or more amino acid residues are bound to the amine group of the amino acid ester.
  • L 2 comprises or consists of: wherein R 1 and R 1' are independently selected from hydrogen or an optionally substituted C1-3 alkyl or are taken together with the atom on which they are attached to form an optionally substituted C 3 -C 7 carbocyclic ring; each R 3 is an optionally substituted group independently selected from hydrogen, C1-6 alkyl, benzyl, -OH, -O(C1-6 alkyl), and -CN; each R c is hydrogen or optionally substituted C 1-6 alkyl; and n is 1, 2, or 3.
  • the one or more amino acids linked to the amine of the amino acid ester comprise L- or D- isomers of amino acid residues.
  • Naturally-occurring amino acid refers to Ala, Asp, Cys, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, Tyr, or citrulline.
  • D- designates an amino acid having the “D” (dextrorotary) configuration, as opposed to the configuration in the naturally occurring (“L-”) amino acids.
  • the amino acids described herein can be purchased commercially (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art.
  • amino acids with non-natural or artificial side chains are linked to the amine of the amino acid ester.
  • the composition of the linker i.e., the peptide sequence of L 2
  • various permutations of amino acids in L 2 could yield conjugates with faster addition and deblocking kinetics.
  • Such linkers could include variations of amino acid identity and number of consecutive amino acids.
  • the one or more amino acids included in the L 2 portion of the linker / bound to the amino acid ester can be selected, for example, to optimize protease binding and ester cleavage.
  • a combinatorial library can be generated to test optimal cleavage activity, amino acids can be chosen based on existing known peptide sequence targets for the protease.
  • the protease comprising esterase activity can recognize the peptide portion of the linker and hydrolyzes the ester group of the amino acid ester of L 2 , resulting in removal of polymerase attached to the nucleotide via the linker, as disclosed herein.
  • a spacer can be used between the nucleotide and the linker, or between the linker and the label. Different lengths of spacers can be used in order to increase L2 availability towards the protease/esterase and increase the efficiency and fidelity of polymerases. Exemplary spacers include, for example, polyethyleneglycol or other suitable spacers.
  • linker structures may include, but are not limited to, carbon-chain linkers (e.g., C6, C12, C18, C24, etc.), peptide linkers (e.g., poly-glycine or poly-alanine ranging from about 1 residue to about 1,000 residues in length), or polyether linkers (e.g., PEG, PPG, PAG, PTMG from about 1 polyether unit to about 1,000 polyether units in length).
  • carbon-chain linkers e.g., C6, C12, C18, C24, etc.
  • peptide linkers e.g., poly-glycine or poly-alanine ranging from about 1 residue to about 1,000 residues in length
  • polyether linkers e.g., PEG, PPG, PAG, PTMG from about 1 polyether unit to about 1,000 polyether units in length.
  • L 1 or L 3 is a chain of atoms selected from C, N, O, S, Si, and P, preferably having 0-500 atoms, wherein L 1 covalently connects to Nuc and L 2 , and wherein L 3 covalently connects to L 2 and Pol.
  • L 1 or L 3 may be combined in all chemically relevant ways, such as forming alkylene, alkenylene, and alkynylene, carbamates, carbonates, ethers, polyoxyalkylene, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids, peptides, acyloxylamines, hydroxamic acids, or combination above thereof.
  • L 1 or L 3 comprises one or more carbon atoms, zero, one, or more oxygen atoms, zero, one or more nitrogen atoms, zero, one, or more sulfur atoms, or a combination thereof, in different embodiments.
  • L 1 or L 3 comprise, comprise about, comprise at least, comprise at least about, comprise at most, or comprise at most about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000
  • L 1 or L 3 comprise a polymer, such as a homopolymer or a heteropolymer. In some embodiments, L 1 or L 3 comprise a plurality of repeat units. In some embodiments, the plurality of repeating units comprises identical repeating units. In some embodiments, the plurality of repeating units comprises two or more different repeating units.
  • the plurality of repeating units can comprise a polyether such as paraformaldehyde, polyethylene glycol (PEG), polypropylene glycol (PPG), polyalkylene glycol (PAG), polytetramethylene glycol (PTMG), or a combination thereof.
  • the plurality of repeating units can comprise PEGix, PEG23, PEG24, or a combination thereof.
  • the plurality of repeating units can comprise a polyalkylene, such as polyethene, polypropene, polybutene, or a combination thereof.
  • a repeating unit of the plurality of repeating units comprises no aromatic group.
  • a repeating unit of the plurality of repeating units comprises one or more aromatic groups.
  • L 1 or L 3 comprises any number of basic chemical starting blocks.
  • linkers may comprise linear or branched alkyl, alkenyl, or alkynyl chains, or combinations thereof, that provide a useful distance between the nucleotide and polymerase, the nucleotide and L 2 , or the polymerase and L 2 .
  • amino-alkyl linkers e.g., amino-hexyl linkers, have been used to attach linkers to nucleotide analogs, and are generally sufficiently rigid to maintain such distances.
  • the longest chain of such linkers may include as many as 2 atoms, 3 atoms, 4 atoms, 5 atoms, 6 atoms, 7 atoms, 8 atoms, 9 atoms, 10 atoms, or even 11-35 atoms, or even 35-50 atoms.
  • the linear or branched linker may also contain heteroatoms other than carbon, including, but not limited to, oxygen, sulfur, phosphate, and nitrogen.
  • a polyoxyethylene chain also commonly referred to as polyethyleneglycol, or PEG is a preferred linker constituent due to the hydrophilic properties associated with polyoxyethylene. Insertion of heteroatom such as nitrogen and oxygen into the linkers may affect the solubility and stability of the linkers.
  • the linker including L 1 or L 3 , may be rigid in nature or flexible.
  • Rigid structures include laterally rigid chemical groups, e.g., ring structures such as aromatic compounds, multiple chemical bonds between adjacent groups, e.g., double or triple bonds, in order to prevent rotation of groups relative to each other, and the consequent flexibility that imparts to the overall linker.
  • the degree of desired rigidity may be modified depending on the content of the linker, or the number of bonds between the individual atoms comprising the linker.
  • addition of ringed structures along the linker may impart rigidity.
  • Ringed structures may include aromatic or non-aromatic rings. Rings may be anywhere from 3 carbons, to 4 carbons, to 5 carbons or even 6 carbons in size.
  • Rings may also optionally include heteroatoms such as oxygen or nitrogen and also be aromatic or non-aromatic. Rings may additionally optionally be substituted by other alkyl groups and/or substituted alkyl groups.
  • Linkers that comprise ring or aromatic structures can include, for example aryl alkynes and aryl amides. Other examples of the linkers of the disclosure include oligopeptide linkers that also may optionally include ring structures within their structure.
  • L 1 or L 3 is a C1-C10 alkylene chain, wherein 1-6 methylene units are optionally and independently replaced by -NH-, -O-, -C(O)-, -C(O)NH-, -NHC(O)-, - NHC(O)NH-, -C(O)O-, -OC(O)-, -SS-, optionally substituted cycloalkylene (e.g., C 3 -C 8 , C 3 - C6, or C5-C6), optionally substituted heterocycloalkylene (e.g., 3 to 8, 3 to 6, or 5 to 6 membered), optionally substituted arylene (e.g., C6-C10, C10, or phenylene), or substituted or unsubstituted heteroarylene (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).
  • cycloalkylene e.g., C 3 -C 8 , C 3
  • L 1 or L 3 is a bond, -NH-, -O-, -C(O)-, -C(O)NH-, -NHC(O)-, - NHC(O)NH-, -C(O)O-, -OC(O)-, -SS-, optionally substituted alkylene (e.g., C1- C20, C10- C 20 , C 1 -C 8 , C 1 -C 6 , or C 1 -C 4 ), optionally substituted heteroalkylene (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), optionally substituted cycloalkylene (e.g., C3-C8, C3- C6, or C5-C6), optionally substituted heterocycloalkylene (e.g., 3 to 8, 3 to 6, or 5 to 6 membered), optionally substituted (e.g., C 6 -C 10 ,
  • alkylene e
  • L 1 or L 3 is optionally substitutedC1-C20 alkylene. In some embodiments, L 1 or L 3 optionally substituted 2 to 20 membered heteroalkylene. In embodiments, L 1 or L 3 is optionally substituted C3-C8 cycloalkylene. In some embodiments, L 1 or L 3 is optionally substituted 3 to 8 membered heterocycloalkylene. In embodiments, L 1 or L 3 is optionally substituted C6-C10 arylene. In embodiments, L 1 or L 3 is optionally substituted 5 to 10 membered heteroarylene. [0419] In some embodiments, L 1 is substituted with 1-6 instances of R L . In some embodiments, L 3 is substituted with 1-6 instances of R L .
  • Each R L is independently selected from the group consisting of oxo, halogen, -CCI3, -CBr3, -CF3, -CI3, -CN, -OH, -NH2, - COOH, -CONH 2 , -NO 2 , -SH, -SO 3 H, -SO 4 H, -SO 2 NH 2 , -NHNH 2 , -ONH 2 , -NHC(O)NHNH 2 , -NHC(O)NH 2 , -NHSO 2 H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl 3 , -OCF 3 , -OCBr 3 , - OCI2, -OCHCI2, -OCHBr2, -OCHb, -OCHF2, -N3, optionally substituted alkyl (e.g., C1-C20, 8 to 20, 2 to C 8 , C 3 -C 6 , or
  • L 1 or L 3 is -(CH 2 CH 2 O) b -. In embodiments, L 1 or L 3 is - CCCH2(OCH2CH2)a-NHC(O)-(CH2)c(OCH2CH2)b-. In embodiments, L 1 or L 3 is - CHCHCH2-NHC(O)-(CH2)c(0CH2CH2)b-. In embodiments, L 1 or L 3 is -CCCH2-NHC(O)- (CH 2 )c(OCH 2 CH 2 ) b -. In embodiments, L 1 or L 3 is -CCCH 2 -. The symbol a is an integer from 0 to 8. In embodiments, a is 1. In embodiments, a is 0.
  • L1 or L3 is independently a substituted or unsubstituted C1-C4 alkylene or substituted or unsubstituted 8 to 20 membered heteroalkylene. [0421] L1 acts as an attachment point to the nucleotide and includes a hydroxyl terminal group which binds to a portion of L2 during synthesis.
  • L1 is a scar that is enzymatically or chemically cleavable after cleavage of polymerase-nucleotide linker / removal of the L2-L3-pol moiety.
  • L1 is selected from the group consisting of a bond, an optionally substituted C1-12 alkylene chain, C4-C20 polyethylene glycol, an optionally substituted C 2-12 alkenylene chain, and an optionally substituted C 2-12 alkynylene chain, wherein 1-4 methylene units of L 1 are optionally and independently replaced with -O-, - N(R b )-, -C(O)-, -S-, -S(O)-, -S(O)2-, phenylene, cyclopropylene; wherein each R b is independently hydrogen or optionally substituted C1-6 alkyl.
  • L1 comprises: wherein each R a is independently selected from the group consisting of halogen, hydroxyl, cyano, optionally substituted C1-6 alkyl, and optionally substituted C1-6 alkoxy. [0425] In some embodiments, L 1 is selected from the group consisting of: . [0426] In some embodiments, L3 comprises a bioconjugate group suitable for conjugation of L3 to the polymerase. [0427] In some embodiments, the bioconjugate group is an N-hydroxysuccinimide ester (NHS) group. In some embodiments, the bioconjugate group is a maleimide group.
  • NHS N-hydroxysuccinimide ester
  • the linker may then be covalently attached to the polymerase by reaction of the maleimide group with a cysteine residue of the polymerase.
  • the polymerase may be operably linked to a linker moiety including a covalent or non-covalent bond; amino acid tag (e.g., poly-amino acid tag, poly- His tag, 6His-tag (SEQ ID NO: 53)); chemical compound (e.g., polyethylene glycol); protein-protein binding pair (e.g., biotin-avidin); affinity coupling; capture probes; or any combination of these.
  • the linker moiety can be separate from or part of a polymerase variant.
  • the linker connecting the nucleotide and the polymerase comprises a saturated or unsaturated, substituted, or unsubstituted, straight, or branched carbon chain.
  • the length of the linker can be different in different embodiments. The length of the linker may vary depending on the type of nucleotide and the polymerase. In some embodiments, the linker length in the enzyme linked nucleotide is different for each different nucleotide or nucleotide analog.
  • the linker has a length of, of about, of at least, of at least about, of at most, or of at most about, 19 ⁇ , 20 ⁇ , 21 ⁇ , 22 ⁇ , 23 ⁇ , 24 ⁇ , 25 ⁇ , 26 ⁇ , 27 ⁇ , 28 ⁇ , 29 ⁇ , 30 ⁇ , 31 ⁇ , 32 ⁇ , 33 ⁇ , 34 ⁇ , 35 ⁇ , 36 ⁇ , 37 ⁇ , 38 ⁇ , 39 ⁇ , 40 ⁇ , 41 ⁇ , 42 ⁇ , 43 ⁇ , 44 ⁇ , 45 ⁇ , 46 ⁇ , 47 ⁇ , 48 ⁇ , 49 ⁇ , 50 ⁇ , 51 ⁇ , 52 ⁇ , 53 ⁇ , 54 ⁇ , 55 ⁇ , 56 ⁇ , 57 ⁇ , 58 ⁇ , 59 ⁇ , 60 ⁇ , 61 ⁇ , 62 ⁇ , 63 ⁇ , 64 ⁇ , 65 ⁇ , 66 ⁇ , 67 ⁇ , 68 ⁇ , 69 ⁇ , 70 ⁇ ,
  • the distance between the linked atom of the nucleotide and the polymerase is about 5 ⁇ to about 20 ⁇ . In some embodiments, the distance between the linked atom of the nucleotide and the polymerase is about 20 ⁇ to about 50 ⁇ . In some embodiments, the distance between the linked atom of the nucleotide and the polymerase is about 50 ⁇ to about 75 ⁇ . In some embodiments, the distance between the linked atom of the nucleotide and the polymerase is about 75 ⁇ to about 100 ⁇ .
  • the length of the linker will be defined as its persistence length, corresponding to the root-mean-square (RMS) distance between the ends of the linker as characterized by dynamic simulations, 2-D trapping experiments, or ab initio calculations based on statistical distributions of polymers in compact, collapsed, or fluid states as required by the solution, suspension, or fluid conditions present.
  • RMS root-mean-square
  • a linker may have a persistence length of at least 0.1, at least 0.2, at least 0.4, at least 1, at least 2, at least 4, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 700, or at least 1,000 nm, or a persistence length in a range defined by or comprising any two or more of these values.
  • a linker for connecting the nucleotide to the enzyme can have a persistence length of about 0.1 - 1,000 nm, 0.5 - 500 nm, 0.5 - 400 nm, 0.5 - 300 nm, 0.5 - 200 nm, 0.5 - 100 nm, 0.5 - 50 nm, 1 - 500 nm, 1 - 400 nm, 1 - 300 nm, 1 - 200 nm, 1 - 100 nm, 1-50 nm, 1.5 - 500 nm, 1.5 - 400 nm, 1.5 - 300 nm, 1.5 - 200 nm, 1.5 - 100 nm, 1.5 - 50 nm, 5 - 500 nm, 5 - 400 nm, 5 - 300 nm, 5 - 200 nm, 5 - 100 nm, or 5 - 50 nm.
  • the linker may have a persistence length of shorter than about 5, 10, 20, 30, 40, 50, 60, 80, 100, 200, 300, 400, 500, 700, or 1,000 nm.
  • linkers provided for one nucleotide may be longer or shorter than the linker provided for another nucleotide.
  • linkers provided for one polymerase may be longer or shorter than the linker provided for another polymerase.
  • the conjugate is represented by [0432]
  • a conjugate is represented by a structure of Formula (I) or (II): wherein L 1 is selected from the group consisting of an optionally substituted C 1-6 alkylene chain, an optionally substituted C2-6 alkenylene chain, and an optionally substituted C1-6 alkynylene chain, wherein 1-4 methylene units are optionally and independently replaced with -O-, - N(R a )-, -C(O)-, -S-, -S(O)-, -S(O) 2 -, or phenylene;
  • L 2 is a cleavable linker;
  • L 3 is a linker connecting pol to L 2 each R a is independently hydrogen or C 1-6 alkyl;
  • R 2 is hydrogen or methyl;
  • R is a ribose polyphosphate or deoxyribose polyphosphate; and
  • Pol is a polymerase.
  • thermostable polymerases Wild-type template independent polymerases such as Terminal deoxynucleotidyl Transferase (TdT) are not thermostable. Use of bases with exocyclic amines masked as azido groups has also been explored (Nuclera Nucleics PCT Publication WO2020229831A1). However, the unmasking reagent (TCEP) causes DNA damage, and there are doubts about the stability of the azido modification. [0435] In some embodiments, provided herein are improved methods for enzymatic synthesis of long polynucleotides by cyclic stepwise extension using a template-independent polymerase and modified nucleotides to prevent secondary structure formation.
  • TdT Terminal deoxynucleotidyl Transferase
  • nucleobases can be converted back into their native form by removal of the protecting group to facilitate further use and/or downstream processing of the synthesized polynucleotides.
  • nucleobase to inhibit secondary structure during polynucleotide synthesis, which can be efficiently removed after synthesis to leave a polynucleotide without modified nucleotides that might interfere with downstream applications.
  • modified nucleotides are shown herein to be useful for both enzymatic polynucleotide synthesis with free nucleotides and for conjugate-based polynucleotide synthesis.
  • compositions and methods of oligonucleotide synthesis that inhibit secondary structure formation and improve oligonucleotide synthesis length and accuracy by providing protecting groups attached to the nucleobase of the synthesized polynucleotide that inhibit secondary structure formation.
  • these are provided as modified nucleotides incorporated into an oligonucleotide during enzymatic synthesis.
  • these are provided as part of a linker-nucleotide conjugate used during enzymatic oligonucleotide synthesis, such that the linker is attached to a base pairing nitrogen or oxygen atom on the nucleobase, and cleavage of the linker to separate the polymerase from the nucleotide during synthesis leaves a portion of the linker attached to a base pairing nitrogen or oxygen atom, which can act as a protecting group (also referred to herein as a “scar”) to inhibit secondary structure formation, as shown in FIG. 2.
  • a protecting group also referred to herein as a “scar”
  • the present disclosure includes a method of synthesizing a polynucleotide, comprising: providing a polynucleotide comprising one or more protected nucleobases and removing one or more protecting groups from said protected nucleobases.
  • providing a polynucleotide comprises: contacting a precursor polynucleotide with a nucleotide comprising a protected nucleobase and a template- independent polymerase; and adding said nucleotide to the 3′ end of said precursor polynucleotide via said template-independent polymerase.
  • a method of synthesizing a polynucleotide further comprises repeating contacting and adding step one or more times.
  • a “protected” nucleotide refers to a nucleotide that has biomolecule attached to a base pairing oxygen or nitrogen on the nucleobase.
  • the biomolecule inhibits hydrogen bonding of the oxygen or nitrogen to other nucleotides, such as in Watson-Crick base pairing, G-quadruplex formation, and other types of hydrogen bonding that can generate secondary structure.
  • the biomolecule inhibits formation of secondary structure during oligonucleotide synthesis.
  • a “scarred” nucleotide and a “protected” nucleotide can both refer to the same structure when a linker is bound to an oxygen or nitrogen on the nucleobase. Such that cleavage of the linker leaves a “scarred” nucleotide that is also a “protected” nucleotide.
  • a conjugate linker is attached to a base pairing oxygen or nitrogen to take advantage of the presence of a scar to provide a protected nucleotide to inhibit secondary structure formation during synthesis.
  • Protected nucleotides can also refer to modified nucleotides using during oligonucleotide synthesis that are not part of a conjugate, but are useful to prevent secondary structure during oligonucleotide synthesis.
  • the present disclosure includes a method of synthesizing a polynucleotide comprising contacting a precursor polynucleotide with a nucleotide and a polymerase, wherein said nucleotide comprises comprising a protecting group bound to a base pairing oxygen or nitrogen on the nucleobase.
  • the method of synthesizing a polynucleotide comprises removing a blocking group, such as a conjugated polymerase or a reversible terminator, after addition of a nucleotide to a precursor polynucleotide.
  • a blocking group such as a conjugated polymerase or a reversible terminator
  • the method of synthesizing a polynucleotide comprises repeating contacting, adding, and optionally removing a blocking group described herein one or more times.
  • removal of one or more protecting groups described herein comprises exposing said polynucleotide to a chemical or photolytic condition capable of removing said one or more protecting groups from said protected nucleobases.
  • synthesis can be completed entirely with protected nucleotides, synthesis with a combination of unmodified and protected nucleotides can also be used effectively to improve polynucleotide synthesis.
  • only one of the four nucleotides added e.g., from G or T
  • protected nucleotides are only added at targeted positions where secondary structure or ternary structure is predicted, which could interfere with synthesis.
  • Such structures can be predicted based on the presence of complementary DNA regions in various ways and respective tools exist, such as the NUPACK algorithms (http://www.nupack.org/home/model).
  • synthesis of a completed polynucleotide where synthesis is improved can include the use of only 1 or 2 protected nucleotides.
  • about 5%, about 10%, about 20%, about 30%, about 50%, substantially all, or 100% of a specific nucleotide is incorporated into the polynucleotide in their protected version.
  • less than 5%, less than 10%, less than 20%, less than 30%, or less than 50% of a specific nucleotide is incorporated into the polynucleotide in its protected version.
  • nucleotide synthesis is performed such that the last and first 1, 2, or 3 positions of the synthesized nucleic acid does not comprise protected nucleotides.
  • the present disclosure includes a method of synthesizing a polynucleotide comprising contacting a precursor polynucleotide with a conjugate comprising a nucleotide covalently linked to a polymerase via a cleavable linker, wherein cleavage of said nucleotide from said polymerase generates a scarred nucleobase.
  • the method of synthesizing a polynucleotide comprises cleaving a cleavable linker after addition of a nucleotide to a precursor polynucleotide.
  • the method of synthesizing a polynucleotide comprises repeating contacting, adding, and optionally cleaving steps described herein one or more times.
  • removal of one or more scars described herein comprises contacting said polynucleotide with a chemical of photolytic condition capable of removing said one or more scars from said scarred nucleobases.
  • synthesis of a polynucleotide comprises adding nucleotides stepwise to a starter molecule (e.g., an initial oligonucleotide) via the cycled steps of: addition of polymerase-nucleotide conjugate to an oligonucleotide, binding of the nucleotide to the 3′ end of the oligonucleotide catalyzed by the polymerase, and cleavage of the polymerase from the added nucleotide. These steps can be repeated until a desired polynucleotide is synthesized.
  • a starter molecule e.g., an initial oligonucleotide
  • nucleotides comprising protected nucleobases helps to improve the efficiency and accuracy of synthesis by inhibiting secondary structure formation which can interfere with the addition of the incoming nucleotide by the polymerase during synthesis.
  • a portion of the linker may remain attached to the nucleotide, leaving a scar as compared to a naturally occurring nucleotide. This can negatively impact downstream use the synthesized polynucleotide, including amplification of the synthesized polynucleotide, or direct use for its intended application.
  • One advantage of the methods and compositions provided herein is that they allow scarless synthesis of a polynucleotide when using polymerase-nucleotide conjugates for synthesis.
  • Preferred linker structures and methods of removing residual scars / protecting groups after oligonucleotide synthesis to leave a naturally occurring polynucleotide without scars are also provided herein.
  • a “scarred” nucleotide refers to a nucleotide that has a portion of a linker still attached to the nucleotide after cleavage of the linker to release an attached biomolecule, such as a polymerase of a polymerase-nucleotide conjugate.
  • an attached biomolecule such as a polymerase of a polymerase-nucleotide conjugate.
  • the resulting polynucleotide can then be treated suitable conditions as described herein to remove the scar from the modified nucleotides, resulting in a polynucleotide with unmodified nucleobases.
  • removal of one or more scars from the synthesized polynucleotide comprises contacting the polynucleotide with suitable conditions capable of removing said one or more scars from a scarred nucleobase.
  • described herein is a method of synthesizing a polynucleotide, comprising: providing a polynucleotide comprising one or more scarred nucleobases and removing one or more scars from said scarred nucleobases.
  • providing a polynucleotide comprises: contacting a precursor polynucleotide with a nucleotide comprising a nucleobase linked to a template-independent polymerase; and adding said nucleotide to the 3′ end of said precursor polynucleotide via said template- independent polymerase.
  • a method of synthesizing a polynucleotide further comprises repeating contacting and adding step one or more times.
  • Enzymatically Removeable Protecting Groups [0449]
  • modified nucleotides or nucleotide- linkers than when the linker is cleaved, a protecting group or scar remains on the nucleobase that can be removed enzymatically.
  • improved methods for synthesis of nucleic acids by cyclic extension using a template-independent polymerase are also provided herein.
  • nucleobases can be converted back into their native form by enzymatic removal of the alkyl group.
  • the present disclosure includes a method of synthesizing a polynucleotide comprising one or more alkylated nucleobases, and removing one or more alkyl groups from said alkylated nucleobases.
  • providing a polynucleotide comprises: contacting a precursor polynucleotide with a nucleotide comprising an alkylated nucleobase and a template-independent polymerase; and adding said nucleotide to the 3′ end of said precursor polynucleotide via said template-independent polymerase.
  • a method of synthesizing a polynucleotide further comprises repeating contacting and adding step one or more times.
  • the resulting polynucleotide can then be treated with an alkyl transferase to remove the alkyl group bound to the nucleotides, resulting in a polynucleotide with unmodified nucleobases.
  • removal of one or more alkyl groups from the synthesized polynucleotide comprises contacting the polynucleotide with one or more enzymes capable of removing said one or more alkyl groups from an alkylated nucleobase.
  • the enzyme suitable for de-alkylation of the alkylated nucleobase is an alkyl transferase.
  • a suitable enzyme for de-alkylating the polynucleotide is an alkyl transferase from EC 2.1.1.63.
  • the alkyl transferase is an AGT (alkylguanine transferase) enzyme, e.g., O 6 -alkylguanine DNA alkyl transferase.
  • the enzyme used to remove the alkyl group from the alkylated nucleobase is AlkB (E.
  • FIG. 3 and FIG. 4 show a reaction including cleavage of a linker in a nucleotide-TdT conjugate and removal of an alkyl group from an alkylated nucleotide by an AGT enzyme.
  • the linker is separate from the alkyl modification of the nucleotide.
  • the TdT is linked to the alkyl modification of the nucleotide and the alkyl group remains after cleavage of the linker binding the nucleotide and the TdT.
  • Suitable enzymes for use in de-alkylating alkylated nucleobases after completion of synthesis can be determined by screening a set of enzymes known to be involved in a de- alkylation reaction of a nucleobase or closely related reaction.
  • One example of an easy screening method is described herein in Example 8. Using such screening methods, one of ordinary skill in the art can identify suitable enzymes for de-alkylation and implementations of this DNA synthesis strategy.
  • O 6 -alkylguanine DNA alkyltransferase and AlkB are exemplified enzymes suitable for de-alkylation
  • a number of other enzymes from various species are closely related and could also be suitable for use in the de-alkylation of the synthesized nucleotides.
  • Tables 1 and 2 below provides a list of alkyl transferases that could be suitable to de-alkylate polynucleotide synthesis products described herein.
  • Table 1 – Alkyl transferase enzyme list wild type
  • R 2 is selected from the group consisting of hydrogen or C1-C2 alkyl optionally substituted with -OH.
  • an alkylated nucleobase is selected from the group consisting of:
  • the linker is attached to a different position of the base than the alkylation.
  • the linker is specifically attached to an amino acid of the polymerase. In these cases, it is preferable to attach the linker to an amino acid at a position that can be mutated without loss of the polymerase activity, e.g., positions 180, 188, 253 or 302 of murine TdT (numbering as in the crystal structure PDB ID: 4127). It is preferable to not attach the linker to an amino acid involved in the catalytic activity of the polymerase to avoid interfering with catalysis.
  • a linker of a conjugate may be attached to the 7-position of deaza dGTP or the 5-position of dTTP or dUTP.
  • the linker of the conjugate is cleaved to leave a scar.
  • a scar may remain on the DNA after cleavage.
  • a scar comprises a hydroxyl.
  • a scar comprises an amine.
  • a scar comprises a hydroxylalkyl group.
  • the linker of a conjugate is attached to an O-alkylated nucleobase at the alkyl group.
  • the linker of a conjugate attached to an O-alkylated nucleobase is cleaved to leave a scar.
  • a scar is removed using an enzyme capable of removing said one or more alkyl groups from an alkylated nucleobase.
  • a ribose polyphosphate is selected from the group consisting of ribose triphosphate, ribose tetraphosphate, ribose pentaphosphate, and ribose hexaphosphate.
  • a ribose polyphosphate is a ribose triphosphate.
  • a ribose polyphosphate is a ribose hexaphosphate.
  • ribose phosphate is a pentaphosphate.
  • ribose polyphosphate is a ribose tetraphosphate.
  • the present disclosure includes a method of treating a polynucleotide synthesized with alkylated nucleobases, comprising: providing a polynucleotide comprising one or more alkylated nucleobases; and removing one or more alkyl groups from said one or more alkylated nucleobases.
  • the present disclosure includes a method of synthesizing a polynucleotide comprising an alkylated nucleobase, comprising: contacting a precursor polynucleotide with a polymerase and a nucleotide comprising said alkylated nucleobase; adding said nucleotide to the 3' end of said precursor polynucleotide via said polymerase.
  • the present disclosure includes a method of synthesizing a polynucleotide comprising contacting a precursor polynucleotide with a conjugate comprising a nucleotide covalently linked to a polymerase via a cleavable linker, wherein said nucleotide comprises said alkylated nucleobase.
  • the method of synthesizing a polynucleotide comprises cleaving a cleavable linker after addition of a nucleotide to a precursor polynucleotide.
  • the method of synthesizing a polynucleotide comprises repeating contacting, adding, and optionally cleaving steps described herein one or more times.
  • removal of one or more alkyl groups described herein comprises contacting said polynucleotide with an enzyme capable of removing said one or more alkyl groups from said alkylated nucleobases.
  • a method of synthesizing a polypeptide comprising contacting said polynucleotide with two or more enzymes capable of removing said one or more alkyl groups from said alkylated nucleobases.
  • synthesis of a polynucleotide comprises adding nucleotides stepwise to a starter molecule (e.g., an initial oligonucleotide) via the cycled steps of: addition of polymerase-nucleotide conjugate to an oligonucleotide, binding of the nucleotide to the 3’ end of the oligonucleotide catalyzed by the polymerase, and cleavage of the polymerase from the added nucleotide. These steps can be repeated until a desired polynucleotide is synthesized.
  • a starter molecule e.g., an initial oligonucleotide
  • nucleotides comprising alkylated nucleobases helps to improve the efficiency and accuracy of synthesis by inhibiting secondary structure formation which can interfere with the addition of the incoming nucleotide by the polymerase during synthesis.
  • synthesis can be completed entirely with alkylated nucleotides, synthesis with a combination of unmodified and alkylated nucleotides can also be used effectively to improve polynucleotide synthesis.
  • only one of the four nucleotides added e.g., from G or T
  • alkylated nucleotides are only added at targeted positions where secondary structure or ternary structure is predicted, which could interfere with synthesis.
  • Such structures can be predicted based on the presence of complementary DNA regions in various ways and respective tools exist, such as the NUPACK algorithms (http://www.nupack.org/home/model).
  • synthesis of a completed polynucleotide where synthesis is improved can include the use of only 1 or 2 alkylated nucleotides.
  • about 5%, about 10%, about 20%, about 30%, about 50%, substantially all, or 100% of a specific nucleotide is incorporated into the polynucleotide in their alkylated version.
  • less than 5%, less than 10%, less than 20%, less than 30%, or less than 50% of a specific nucleotide is incorporated into the polynucleotide in its alkylated version.
  • nucleotide synthesis is performed such that the last and first 1, 2, or 3 positions of the synthesized nucleic acid does not comprise alkylated nucleotides.
  • the nucleotides analogs described herein comprise a reversible terminator group, such as such as an O- azidomethyl or O-NH2 group on the 3' position of the sugar or an (alpha-tertbutyl-2- nitrobenzyl)oxymethl group on the 5 position of pyrimidines or the 7 position of 7- deazapurines (for an overview see, e.g. Chen et al., Genomics, Proteomics & Bioinformatics 2013 11: 34-40).
  • the nucleotide analog prevents or hinders further elongation once incorporated into a nucleic acid to achieve controlled termination of synthesis.
  • the RTdNTP- polymerase conjugates when used as part of a conjugate, do not rely on the shielding effect to achieve termination, e.g., when a 3' modified RTdNTP is tethered to the polymerase, the linker used may exceed 100 A or 200 A in length.
  • O 6 -alkylguanine-DNA alkyltransferase irreversibly transfers an alkyl group from its substrate, a modified nucleotide.
  • alkyl modified nucleotides useful during synthesis, where the alkyl group can be removed by AGT. In some embodiments, this removal converts a ‘scarred’ nucleotide to a naturally occurring nucleotide or nucleobase in a synthesized oligonucleotide.
  • Several forms of the enzyme can be used considered provided they have similar properties in reacting with an alkyl group substrate (such as human, murine, rat, a chimera, or other species of AGT).
  • the alkyl group refers to any group that can act as a substrate and is removed from the nucleotide by an AGT enzyme.
  • O6-alkylguanine-DNA alkyltransferase also includes variants of a wild-type AGT which may differ by virtue of one or more amino acid substitutions, deletions, or additions, but which still retain the property of transferring a label present on a substrate to the AGT part of the fusion protein.
  • AGT variants may be obtained by chemical modification using techniques well known to those skilled in the art.
  • AGT variants may preferably be produced using protein engineering techniques known to the skilled person and/or using molecular evolution to generate and select new O6-aIkylguanine- DNA alkyltransferases.
  • an alkylated nucleobase is a nucleobase of formula (I) or formula (II): wherein
  • R 1 is selected from the group consisting of Ci-6 alkyl, C2-6 alkenyl, C1-6 alkynyl, and -(CH2)o- 3PI1, wherein R 1 is optionally substituted with 1-6 instances of R la ; each R la is independently selected from halogen, C1-6 alkyl, -(CH2)o-30R lb , -NO2, -N3, - OPO2OH, and -(CH 2 )o-3NHR lb ; and each R lb is independently selected from hydrogen, C1-6 alkyl, -C(O)(Ci-6 alkyl), C1-6 haloalkyl, -C(O)(Ci-6haloalkyl), and -CthOAc;
  • R 2 is hydrogen or methyl
  • R is a ribose polyphosphate or deoxyribose polyphosphate.
  • R 1 is selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, C1-6 alkynyl, and -(CH2)o-3Ph, wherein R 1 is optionally substituted with 1-6 instances of R la .
  • R 1 is selected from the group consisting of C2-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, and -(CH2)o-3Ph, wherein R 1 is optionally substituted with 1- 6 instances of R a .
  • R 1 is selected from the group consisting of C1-3 alkyl, C2-4 alkenyl, C2-4 alkynyl, and -CH2PI1, wherein R 1 is optionally substituted with one instance of R a .
  • R 1 is selected from the group consisting of methyl, ethyl, C4 alkenyl, C4 alkynyl, and -CH2PI1, wherein R 1 is optionally substituted with one instance of R a .
  • R 1 is selected from the group consisting of ethyl, C4 alkenyl, C4 alkynyl, and -CH2PI1, wherein R 1 is optionally substituted with one instance of R a .
  • R 1 is selected from the group consisting of C1-C3 alkyl, wherein R 1 is optionally substituted with 1-3 instances of R a . In some embodiments, R 1 is selected from the group consisting of C1-C3 alkyl, wherein R 1 is optionally substituted with 1-3 instances of R a .
  • R 1 is selected from the group consisting of
  • R 1 is selected from the group consisting of methyl, ethyl, and n-propyl optionally substituted with 1-3 instances of R a .
  • R 1 is methyl
  • the nucleotide is selected from the group consisting of
  • suitable conditions for removal of a protecting group from a protected nucleobase include exposing a protected nucleobase to light.
  • light is ultraviolet light.
  • ultraviolet light has a wavelength of about 365 nm.
  • suitable conditions for removal of a protecting group include treating a protected nucleobase with an acidic oxidizing solution. In some embodiments, suitable conditions for removal of a protecting group include treating a protected nucleobase with a solution of nitrous acid.
  • suitable conditions for removal of a protecting group include treating a protected nucleobase with a reducing agent.
  • a reducing agent is a phosphorous-based reducing agent.
  • a phosphorous-based reducing agent is PPI13 or TCEP.
  • a phosphorous- based reducing agent is TCEP.
  • suitable conditions for removal of a protecting group comprises the step of treating a protected nucleobase with an oxidizing agent.
  • an oxidizing agent is capable of oxidizing a thioether (i.e., sulfide) to a sulfone.
  • an oxidizing agent is selected from the group consisting of H2O2, mCPBA, and TAPC.
  • conditions for removal of a protecting group further comprise an alkaline (or high pH) environment.
  • the alkaline environment comprises a pH of about 8.
  • the alkaline environment is one that has a pH higher than the pH at polynucleotide synthesis.
  • suitable conditions for removal of a protecting group comprises the step of treating a protected nucleobase with a base.
  • a base is selected from the group consisting of NH4OH, KOH, NaOH, KOMe, NaOMe, and KOtBu.
  • suitable conditions for removal of a protecting group comprises the step of treating a protected nucleobase with conditions to remove an allyl group. In some embodiments, suitable conditions for removal of a protecting group comprises the step of treating a protected nucleobase with Pd. In some embodiments, suitable conditions for removal of a protecting group comprises the step of treating a protected nucleobase with Pd(OAc), Pd2(dba)3, and Pd2(pmdba)3.
  • Described herein are removeable protecting group or scar structures that can be chemically removed from a nitrogen or oxygen of a nucleobase, leaving the natural nucleotide.
  • a linker connecting the nucleotide to the polymerase is attached to the nucleotide at a nitrogen or oxygen on the nucleobase, such that cleavage of the linker leaves the removeable protecting group on the nitrogen or oxygen.
  • the protecting group / scar structures described herein remain attached to the nitrogen or oxygen on the nucleobase during synthesis but are removed before downstream processing or use of the newly synthesized oligonucleotide.
  • a nucleotide comprising a removeable protecting group structure described herein bound to a nitrogen or oxygen atom of the nucleobase (with a structure corresponding to a removeable scar after cleavage of a conjugate linker) can be used for oligonucleotide synthesis.
  • the polymerase-nucleotide conjugates comprise a linker attached to the nucleobase represented by the following structure: where Y is a nucleobase; L is a linker attached to a base-pairing nitrogen or oxygen of the nucleobase; R is a ribose polyphosphate or deoxyribose polyphosphate; and Pol is a polymerase.
  • the linker L can be represented by the formula Z-L 1 -L 2 -L 3 , where Z-L '-L 2 represents a portion of the linker that remains after cleavage of the linker to separate the polymerase from the nucleotide, and L 3 is a linker that can be cleaved from L 2 , and which is attached to the polymerase.
  • scarred nucleotides are nucleotides comprising the remainder of a linker, i.e., a “scar” after cleavage of the linker to separate the polymerase from the nucleotide.
  • Protected nucleotides are nucleotides that comprise a protecting group bound to a base pairing oxygen or nitrogen of the nucleobase. The protected nucleotides have a protecting group structure that corresponds with the structure of the scarred nucleotides.
  • the scarred nucleotides or protected nucleotides have a nucleobase represented by the followings structure: where Y is a nucleobase; L-R 1 is a protecting group or scar; L is a moiety attached to a basepairing nitrogen or oxygen of the nucleobase; and R 1 is selected from the group consisting of hydrogen, -OH, - N(R b ) 2 , and -SH, wherein each R b is independently hydrogen or optionally substituted Ci-6 alkyl.
  • L for protected nucleotides / scarred nucleotides can be represented by the formula Z-L'-L 2 , which corresponds to the same Z-L'-L 2 formula in the conjugate structure.
  • Exemplary removeable scars / protecting groups comprising Z-L 1 -L 2 -R 1 and conditions for removal from the nucleotide are also described herein.
  • removal is achieved by exposure of a photocleavable scar or protecting group to the corresponding wavelength of light.
  • removal is achieved by an appropriate set of chemical conditions, such as a basic environment.
  • Z is selected from the group consisting of a bond, -C(O)-, - C(O)CH 2 -, -C(O)C(R L ) 2 -, -C(O)CH(R L )-, -C(O)O-, and -C(O)N(H)-;
  • L 1 is selected from the group consisting of a bond, wherein L 1 is optionally substituted with 1-4 instances of R L ; each R L is independently selected from the group consisting of halogen, hydroxyl, oxo, and optionally substituted Ci- C3 alkyl, wherein 2 instances of R 1 are optionally taken together with the intervening atom(s) to form a 3-6 membered carbocyclyl ring;
  • L 2 is selected from the group consisting of a bond, an optionally substituted C1-12 alkylene chain, C4-C20 polyethylene glycol, an optionally substituted C2-12 alkenylene chain, and an optionally substituted C2-12 alkynylene chain, wherein 1-6 methylene units are optionally and independently replaced with -O-, -N(R b )-, - C(O)-, -S-, -S(O)-, -S(O)2-, or phenylene;
  • W is selected from the group consisting of
  • a conjugate comprising a linker with a removable scar is bound to an oxygen on the nucleobase of the nucleotide.
  • a scar or a protecting group is bound to an oxygen on the nucleobase of the nucleotide.
  • the oxygen is a base pairing oxygen on the nucleobase.
  • the linker, scar or protecting group is bound to the 04 oxygen of uracil or adenine, or to the 06 oxygen of guanine. Examples of chemically removeable scars or protecting groups are described below:
  • a sulfonyl group is used in a protecting group or scar that can be removed from the nucleobase.
  • An O-linked sulfonyl group can be removed by a suitable base, such as NH4OH, in a beta-elimination reaction.
  • a linker of a conjugate attached to an oxygen or nitrogen on the nucleobase comprises a sulfonyl group.
  • a scar remaining after cleavage of the linker comprises a sulfonyl group.
  • a protected nucleotide comprises an O-linked protecting group comprising a sulfonyl group.
  • a scar or protecting group comprising a sulfonyl group is removed from the nucleotide upon exposure to an appropriate base.
  • a suitable base is a strong base.
  • a suitable base is a hydroxide with a suitable counterion.
  • a suitable base is selected from the group consisting of NaOH, NH4OH, KOH, KOtBu, NaOMe, and KOMe.
  • a linker comprising a sulfonyl group.
  • These Z1-L1-L2 structures can also be used for scarred nucleotides (after linker cleavage) or protected nucleotides for synthesis.
  • Exemplary conjugates comprising a sulfonyl group attached to an oxygen on the nucleobase that is retained as a scar on the nucleotide after cleavage of the linker) and can be removed are shown below:
  • the present disclosure includes a method of preparing a polynucleotide comprising treating a scarred nucleobase comprising a sulfonyl group with a suitable base.
  • treatment of a scarred nucleobase comprising a sulfonyl group with a suitable base results in beta-elimination:
  • a conjugate or nucleotide comprising a sulfonyl group can be prepared as outlined in Scheme 1 : [0510]
  • a thioether group is used in a protecting group or scar that can be removed from the nucleobase. A thioether group can be removed by exposure to a suitable nucleophile.
  • a linker of a conjugate attached to an oxygen or nitrogen on the nucleobase comprises a thioether.
  • a scar remaining after cleavage of the linker comprises a thioether.
  • a protected nucleotide comprises an O-linked or N-linked protecting group comprising a thioether.
  • a scar or protecting group comprising a thioether is removed upon exposure to a suitable oxidant followed by exposure to a suitable base.
  • Z1-L1-L2 structures in conjugates with a linker comprising a nitrobenzyl photocleavable group. These Z1-L1-L2 structures can also be used for scarred nucleotides (after linker cleavage) or protected nucleotides for synthesis.
  • conjugates comprising a photocleavable group attached to an oxygen on the nucleobase (that is retained as a scar on the nucleotide after cleavage of the linker) and can be removed are shown below:
  • the present disclosure includes a method of preparing a polynucleotide comprising removal of a scar or protecting group comprising a thioether group and attached to a nucleobase via oxidation to generate a sulfonyl group and treatment with a suitable base to remove the sulfonyl group.
  • removal of a sulfonyl group bound to an oxygen of the nucleobase can be accomplished through exposure to an oxidant followed by exposure to a base as follows:
  • a conjugate or nucleotide comprising a thioethyl can be prepared as outlined in Scheme 2
  • a cyanoethyl group is used in a protecting group or scar that can be removed from the nucleobase.
  • An O-linked cyanoethyl group can be removed by a suitable base, such as NH4OH.
  • a linker comprises a cyanoethyl group.
  • a scar comprises a cyanoethyl group.
  • a scar comprises a cyanoethyl group that is removed upon exposure to an appropriate base.
  • a suitable base is a strong base.
  • a suitable base is a hydroxide with a suitable counterion.
  • a suitable base is selected from the group consisting of NaOH, NH4OH, KOH, KOtBu, NaOMe, and KOMe.
  • a nucleotide is selected from the group consisting of
  • Zl- L1-L2 structures attached to an oxygen of a protected nucleobase with a linker comprising a cyanoethyl group.
  • These Zl- L1-L2 structures can be used for conjugates that leave scarred nucleotides (after linker cleavage) or protected nucleotides for synthesis:
  • a protected or scarred nucleobase is selected from the group consisting of: [0525]
  • the present disclosure includes a method of preparing a polynucleotide comprising treating a scarred nucleobase comprising a cyanoethyl group with a suitable base.
  • treatment of a scarred nucleobase comprising a cyanoethyl group with a suitable base results in beta-elimination:
  • a conjugate or nucleotide comprising a cyanoethyl group can be prepared as outlined in Scheme 3:
  • an allyl group is used in a protecting group or scar that can be removed from the nucleobase.
  • a linker comprises an allyl group.
  • a scar comprises an allyl group.
  • a scar comprises an allyl group that is removed upon exposure to an appropriate transition metal catalyst.
  • an appropriate transition metal catalyst is a palladium catalyst.
  • an appropriate transition metal catalyst is selected from the group consisting of Pd 2 (dba) 3 , Pd 2 pmdba) 3 , PdCl 2 , Pd(TFA) 2 , and Na 2 PdCl 4 .
  • Zl- L1-L2 structures attached to an oxygen of a protected nucleobase with a linker comprising a cyanoethyl group.
  • These Zl- L1-L2 structures can be used for conjugates that leave scarred nucleotides (after linker cleavage) or protected nucleotides for synthesis:
  • the present disclosure includes a method of preparing a polynucleotide comprising treating a scarred nucleobase comprising an allyl group with a transition metal catalyst and, optionally, one or more suitable ligand.
  • a suitable ligand is P(PhSO3Na)3.
  • a conjugate or nucleotide comprising an allyl group can be prepared as outlined in Scheme 4:
  • an azide is used in a protecting group or scar that can be removed from the nucleobase.
  • a linker comprises an azide.
  • a scar comprises an azide.
  • a scar comprises an azide that is removed upon exposure to a suitable reductant.
  • a suitable reductant is a phosphine.
  • a suitable reductant is TCEP.
  • Z1-L1-L2 structures in conjugates with a linker comprising an azide. These Z1-L1-L2 structures can also be used for scarred nucleotides (after linker cleavage) or protected nucleotides for synthesis.
  • Exemplary conjugates comprising a photocleavable group attached to an oxygen on the nucleobase (that is retained as a scar on the nucleotide after cleavage of the linker) and can be removed are shown below:
  • the present disclosure includes a method of preparing a polynucleotide comprising treating a scarred nucleobase comprising an azidyl group with a suitable reductant.
  • treatment of a scarred nucleobase comprising an azidyl group with a suitable reductant results in removal of the scar:
  • an oxime is used in a protecting group or scar that can be removed from the nucleobase.
  • a linker comprises an oxime.
  • a scar comprises an oxime.
  • a scar comprises an oxime that is removed upon exposure to suitable nucleophile and subsequently to a suitable acid.
  • a suitable nucleophile is HoNOtBu.
  • a suitable acid is a HONO.
  • Z1-L1-L2 structures in conjugates with a linker comprising an oxime. These Z1-L1-L2 structures can also be used for scarred nucleotides (after linker cleavage) or protected nucleotides for synthesis.
  • exemplary conjugates comprising an oxime attached to an oxygen on the nucleobase that is retained as a scar on the nucleotide after cleavage of the linker) and can be removed are shown below:
  • the present disclosure includes a method of preparing a polynucleotide comprising treating a scarred nucleobase comprising an oximyl group with a suitable nucleophile and, subsequently a suitable acid.
  • removal of an oximyl group can be accomplished as shown below:
  • a silyl group is used in a protecting group or scar that can be removed from the nucleobase.
  • a linker comprises a silyl group.
  • a scar comprises a trimethylsilyl group.
  • a linker comprises a silyl group.
  • a scar comprises a trimetylsilyl group.
  • a scar comprises a silyl group that is removed upon exposure to a halogen source.
  • a scar comprises a silyl group that is removed upon exposure to ZnBr2.
  • a scar comprises a silyl group that is removed upon exposure to suitable fluoride source.
  • a suitable fluoride source is selected from the group consisting of KF, TBAF, and TBAT.
  • a conjugate comprising a linker with a removable scar is bound to a nitrogen on the nucleobase of the nucleotide.
  • a scar or a protecting group is bound to a nitrogen on the nucleobase of the nucleotide.
  • the nitrogen is a base pairing nitrogen on the nucleobase.
  • the linker, scar or protecting group is bound to the N1 or N2 nitrogen of guanine, the N4 nitrogen of cytosine, or the N6 nitrogen of adenine.
  • Examples of chemically removeable scars or protecting groups are described below:
  • Z is carbamate, which is attached to a nitrogen of the nucleobase.
  • an O-linked protecting group or scar structure described above (-L1 or -L1-L2) can be attached to carbamate. Exposure to conditions for removal of these protecting groups or scars from carbamate as described above also results in removal of the carbamate.
  • O-linked removeable scars / protecting groups described above can also be attached to a nitrogen on the nucleobase by using an intermediate structure (e.g., Z), such as a carbamate.
  • an intermediate structure e.g., Z
  • a carbamate moiety can be attached to a nitrogen on the nucleobase, and the groups / moieties (-L1 or -L1-L2) described above can be attached to the oxygen of the carbamate.
  • the groups / moieties (-L1 or -L1-L2) described above can be attached to the oxygen of the carbamate.
  • Under conditions suitable for removal of these protecting groups or scars from the carbamate also results in release of the carbamate from the nitrogen of the nucleobase.
  • Z1-L1-L2 structures in conjugates with a linker comprising a sulfonyl group bound to a nitrogen atom via a carbamate group. These Zl-Ll- L2 structures can also be used for scarred nucleotides (after linker cleavage) or protected nucleotides for synthesis.
  • Exemplary conjugates comprising a sulfonyl group attached to a nitrogen (via a carbamate) on the nucleobase (that is retained as a scar on the nucleotide after cleavage of the linker) and can be removed are shown below:
  • a cyanoethyl scar / protecting group attached to a carbamate include N-linked carbamates bound to L1-L2 structures having an electron withdrawing group.
  • Z1-L1-L2 structures in conjugates with a linker comprising a thioether group bound to a nitrogen atom via a carbamate group. These Zl-Ll- L2 structures can also be used for scarred nucleotides (after linker cleavage) or protected nucleotides for synthesis.
  • Exemplary conjugates comprising a thioether group attached to a nitrogen (via a carbamate) on the nucleobase (that is retained as a scar on the nucleotide after cleavage of the linker) and can be removed are shown below:
  • a linker comprises an acyl group linked to a nitrogen of a nucleobase.
  • a scar or protecting group comprises an acyl group linked to a nitrogen of a nucleobase.
  • Z is an acyl group.
  • the scar or protecting group comprising an acyl group is removed upon exposure to an appropriate nucleophile or base.
  • a scar comprises a group that is removed upon exposure to an appropriate nucleophile or base.
  • the acyl group is a carbamate or amide.
  • a linker comprises a carbamate group.
  • a scar comprises a carbamate group.
  • a scar comprises a carbamate group that is removed upon exposure to an appropriate base. In some embodiments, a scar comprises a group that is removed upon exposure to an appropriate base.
  • a suitable base is a strong base. In some embodiments, a suitable base is a hydroxide with a suitable counterion. In some embodiments, a suitable base is selected from the group consisting of NaOH, NH 4 OH, KOH, KOtBu, NaOMe, and KOMe.
  • -Z-L 1 -L 2 -R 1 is where X is O, CH2, or NH; n is 1 or 2; and R 1 is hydrogen, -OH, - N(R b )2, and -SH, wherein each R b is independently hydrogen or optionally substituted C 1-6 alkyl; and wherein the hydrogen atoms bound to the carbon atoms between the carbonyl and the R1 group are each optionally substituted with a C1-3 alkyl group.
  • -Z-L 1 -L 2 -R 1 is selected from the group consisting of , [0564]
  • the results from Example 11 suggest that exposure of a scarred or protected nucleobase comprising a carbamate or amide group linked to an alkyl group of a preferred length results in the cyclization and subsequent removal of the scar, which is more preferred than the nucleophilic or base removal mechanism described above.
  • Example 12 the results from Example 12 suggest that removal of a scar via intramolecular cyclization can be improved by exploiting the Thorpe-Ingold Effect.
  • the introduction of substituent groups on L 1 can increase the rate of removal of a scar.
  • a scar comprising single methyl substituent may be removed faster than an unsubstituted scar.
  • Exemplary intramolecular cyclization reactions for removal of a scar or protecting group comprising an unsubstituted and substituted alkyl group are shown in FIG. 5 A (alkyl group attached to an amide group linked to a nitrogen on the nucleobase), and FIG. 5B (alkyl group attached to a carbamate group linked to a nitrogen on the nucleobase.
  • alkyl substituents occur on carbon groups at any position between the carbonyl group and the R 1 group, except for at the carbon next to R 1 .
  • -Z-L 1 -L 2 -R 1 is selected from the group consisting of
  • a conjugate is selected from the group consisting of
  • a scarred nucleobase is selected from the group consisting of
  • a conjugate or nucleotide comprising carbamate or amide (Z) bound to a nitrogen of a nucleotide and linked to an L1-L2-R1 or an L1-L2-L3 can be prepared as outlined below:
  • molecular sieves and three small round bottom flasks were placed in a drying oven for at least 16 hr. Two small flasks from the oven were charged with molecular sieves and flame-activated under vacuum. While these were cooling, the other small flask was attached to a Hickman distillation apparatus and flame dried. Upon cooling, the first two flasks were backfilled with nitrogen. Trimethyl phosphate and tributyl amine were then placed over the molecular sieves in the initial two flasks for drying. The Hickman distillation apparatus was then used to freshly distill POCI3. The vacuum desiccator was purged with N2 gas, and the flasks inside were then transferred to nitrogen balloons or the Schlenk line.
  • Trimethyl phosphate (40 eq) was added to the nucleoside and the mixture was cooled at -5 °C.
  • To this nucleoside mixture was added dry tributyl amine (3 eq) followed by POCI3 (2.1 eq) slowly via micro syringe. The combined mixture was stirred at -5 °C. for 45 mins. After 45 min, the reaction mixture was treated with a mixture of tributylamine pyrophosphate (5 eq, 0.5 M in dry acetonitrile) and tributyl amine (6 eq). After 1 hour, the mixture was treated with triethylammonium bicarbonate (0.5 M, 1:2 of the total reaction volume) and allowed to stir at ambient temperature for 1 hour.
  • Fmoc Deprotection (compound 5/10/32/33/34): Then, this reaction mixture was further treated with N-Methyl piperidine (1/5 111 of total reaction volume) and stirred for 90 mins at ambient temperature followed by extraction with dichloromethane (2X). The aqueous layer was then purified by reverse phase HPLC (0.1 M triethylammonium acetate buffer/Acetonitrile, 4-47%, 0-15 min, flow 5 ml min' 1 ). Product containing fractions were pooled and lyophilized to provide desired product as a triethylammonium salt. The resulting solid was reconstituted in RNase Free DI water for further experiments.
  • Scheme 11 General synthetic scheme for the access of cleavable amide and carbamate modified nucleotides attached to the N2 positions of G:
  • Scheme 12 General synthetic scheme for the access of cleavable amide and carbamate modified nucleotides attached to the N1 position of G:
  • Scheme 13 General synthetic scheme for the access of cleavable amide and carbamate modified nucleotides attached to the N6 position of A
  • Scheme 14 General synthetic scheme for the access of cleavable amide and carbamate modified nucleotides attached to the N4 position of C
  • Scheme 15 General synthetic scheme for the access of cleavable amide and carbamate modified nucleotides attached to the N3 position of U/T
  • a photocleavable group is used in a protecting group or scar that can be removed from the nucleobase.
  • a photocleavable group can be removed by exposure to appropriate wavelength(s) of light to leave a native nucleotide.
  • the wavelength of light is ultraviolet light (uv).
  • a linker of a conjugate attached to an oxygen or nitrogen on the nucleobase comprises a photocleavable group.
  • a scar after cleavage of the linker comprises a photocleavable group.
  • a nucleotide comprises a protecting group comprising a photocleavable group.
  • L 1 comprises a photocleavable group. In some embodiments, L 1 comprises an optionally substituted 2-nitrobenzyl group. In some embodiments, L 1 comprises a 5-methoxy-2-nitrobenzyl group. In some embodiments, L 1 is wherein, each R a is independently selected from the group consisting of halogen, -Me, and - OMe.
  • L 1 is
  • a conjugate comprising a nucleotide bound to a photocleavable group is represented by: wherein the linker is attached to the nucleotide at an oxygen or nitrogen of the nucleobase.
  • a protected nucleotide or scarred nucleotide comprising a nucleotide bound to a photocleavable group is represented by: wherein the nitrobenzyl group is attached to the nucleotide at an oxygen or nitrogen of the nucleobase.
  • Z1-L1-L2 structures in conjugates with a linker comprising a nitrobenzyl photocleavable group. These Z1-L1-L2 structures can also be used for scarred nucleotides (after linker cleavage) or protected nucleotides for synthesis.
  • conjugates comprising a photocleavable group attached to an oxygen on the nucleobase (that is retained as a scar on the nucleotide after cleavage of the linker) and can be removed are shown below:
  • a protected or scarred nucleotide comprising a photocleavable group is selected from the group consisting of: .
  • a protected nucleobase comprising a photocleavable protecting group or scar bound to an oxygen on the nucleobase is selected from the group consisting of: wherein R 1 is selected from the group consisting of hydrogen, -OH, - N(Rb )2, and -SH; and each Rb is independently hydrogen or optionally substituted C1-6 alkyl.
  • a protected nucleobase is selected from the group consisting of: , ,
  • the present disclosure includes a method of preparing a polynucleotide comprising removal of a scar or protecting group attached to a nucleobase via photolysis.
  • photolysis comprises exposure of a polynucleotide to ultraviolet light.
  • photolysis comprises expose of a polynucleotide to light with a wavelength of about 365 nm.
  • a conjugate or nucleotide comprising a photocleavable group can be prepared as outlined in Scheme 16: Scheme 16 [0596]
  • the photocleavable linker can also be attached to a nitrogen of a nucleobase.
  • An exemplary scheme for preparing an N-linked photocleavable group attached to adenine is outlined in Scheme 17: [0597] Examples of conjugates comprising N-linked carbamate + photocleavable nitrobenzyl protecting groups are shown below.
  • a linker of a conjugate may be attached to an oxygen or nitrogen of the nucleobase.
  • the linker of a conjugate attached to an oxygen or nitrogen of the nucleobase is cleaved to leave a scar, which can also be a protecting group that inhibits secondary structure formation.
  • a scar is removed using a chemical or photolytic condition capable of removing said one or more protecting groups from a protected nucleobase.
  • L3 comprises a bioconjugate group suitable for conjugation of L3 to the polymerase.
  • the bioconjugate group is an N-hydroxysuccinimide ester (NHS) group.
  • the bioconjugate group is a maleimide group. The linker may then be covalently attached to the polymerase by reaction of the maleimide group with a cysteine residue of the polymerase.
  • the polymerase may be operably linked to a linker moiety including a covalent or non-covalent bond; amino acid tag (e.g., poly-amino acid tag, poly- His tag, 6His-tag (SEQ ID NO: 53)); chemical compound (e.g., polyethylene glycol); protein-protein binding pair (e.g., biotin-avidin); affinity coupling; capture probes; or any combination of these.
  • the linker moiety can be separate from or part of a polymerase variant. Phosphatase-treatment of conjugates to reduce insertions [0602]
  • the present disclosure provides polymerase nucleotide conjugates.
  • polymerases can erroneously catalyze covalent addition of nucleotides, which may result in the addition of more than one nucleotide per step when using polymerase-nucleotide conjugates in a controlled, step-wise nucleic acid synthesis (e.g., insertion or non-termination).
  • Technologies provided herein, including combining such conjugates with phosphatases (e.g., providing a polymerase-nucleotide conjugate in the presence of a phosphatase) overcome such challenges.
  • the conjugates are provided in the presence of a phosphatase.
  • the present disclosure provides a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates, wherein a polymerase and a nucleotide are linked via a linker.
  • the linker is cleavable.
  • the conjugate reagent exists in the presence of phosphatases.
  • the polymerase-nucleotide conjugates are combined with a template (e.g., a start oligo or initial oligonucleotide) in the presence of a phosphatase.
  • a template e.g., a start oligo or initial oligonucleotide
  • a typical process for stepwise synthesis of a polynucleotide comprises adding individual nucleotides step-wise to a starter oligo (i.e., an initial oligonucleotide) via cyclical steps.
  • the steps comprise: addition of a polymerase- nucleotide conjugate to an oligonucleotide, covalent addition of the nucleotide to the 3′ end of the oligonucleotide catalyzed by the polymerase, and cleavage of the polymerase from the added nucleotide.
  • steps can be repeated until a desired elongated polynucleotide is synthesized such that the elongated polynucleotide has a length one or more nucleotides longer than the polynucleotide prior to the steps being repeated one or more times.
  • a method of nucleic acid synthesis comprises a step of contacting (e.g., incubating) a conjugate reagent comprising polymerase-nucleotide conjugates (e.g., a plurality of polymerase-nucleotide conjugates) in the presence of one or more phosphatases.
  • a conjugate reagent comprising polymerase-nucleotide conjugates (e.g., a plurality of polymerase-nucleotide conjugates) in the presence of one or more phosphatases.
  • the nucleotides in the plurality are the same nucleotides (e.g., A, G, T, or C, etc.).
  • the nucleotides are different nucleotides (e.g., A, G, T, and/or C, etc.)
  • synthesis conducted in the presence of a phosphatase is improved in one or more ways (e.g., more precise, more efficient, more accurate) as compared to the same synthesis in the absence of a phosphatase.
  • the synthesis performed in the presence of a phosphatase prevents addition of unshielded nucleotides to a nucleic acid.
  • the methods provided herein comprise a step of contacting (e.g., incubating) a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates with a phosphatase, wherein there is a reduction in rates of processes that lead to addition of more than one nucleotide per step when using polymerase- nucleotide conjugates in nucleic acid synthesis (e.g. non-termination leading to an additional nucleotide insertion) as compared to synthesis without phosphatase or without treatment of the conjugate reagent with phosphatase.
  • stepwise nucleic acid synthesis using polymerase-nucleotide conjugates may be susceptible to insertions and/or non-termination resulting in the addition of more than one nucleotide to a nucleic acid in a single step of a cyclic nucleotide extension.
  • An unshielded nucleotide is not sterically-hindered or is only partially sterically- hindered by a tethered polymerase from phosphatase cleavage at its 5′ phosphate.
  • a polymerase can erroneously catalyze the covalent addition of the unshielded nucleotide, which may result in the addition of more than one nucleotide per step when using polymerase-nucleotide conjugates in nucleic acid synthesis (e.g., insertion or non-termination).
  • Technologies provided herein help overcome this challenge to achieve accurate and precise stepwise addition with reduced errors as compared to previously described synthesis approaches.
  • non-termination may occur when an unshielded nucleotide with an uncleaved 5′ phosphate is added to an oligonucleotide.
  • a phosphatase hydrolyzes a 5’ phosphate (e.g., a terminal 5’ phosphate) of a nucleotide (e.g., of a nucleotide triphosphate, etc.).
  • the terminal 5’ phosphate is on an ⁇ -phosphate, ⁇ -phosphate, ⁇ -phosphate, ⁇ -phosphate, ⁇ -phosphate, ⁇ - phosphate, or ⁇ -phosphate of the nucleotide.
  • a phosphatase as disclosed herein hydrolyzes a 5′ phosphate (e.g., a terminal 5’ phosphate) of a nucleotide in a polymerase-nucleotide conjugate or of a free nucleotide.
  • a phosphatase as disclosed herein hydrolyzes 5’ phosphates of nucleotides, e.g., one or more 5′ terminal phosphate(s) of nucleotides in a plurality of polymerase-nucleotide conjugates, and prevents the hydrolyzed nucleotide from addition to the nucleic acid during oligonucleotide synthesis.
  • a phosphatase hydrolyzes a 5′ phosphate of an unshielded nucleotide of a polymerase-nucleotide conjugate. In some such embodiments, the hydrolysis of the 5’ phosphate prevents the unshielded nucleotide from addition to the nucleic acid during oligonucleotide synthesis. In some embodiments, a phosphatase as disclosed herein hydrolyzes one or more 5′ phosphate(s) of unshielded nucleotides in a plurality of polymerase-nucleotide conjugates and prevents said unshielded nucleotides from addition to the nucleic acid during oligonucleotide synthesis.
  • a phosphatase as disclosed herein hydrolyzes one or more 5′ phosphate(s) of one or more free nucleotides in a composition comprising one or more polymerase-nucleotide conjugates and prevents the free nucleotides from addition to the nucleic acid during oligonucleotide synthesis.
  • a phosphatase hydrolyzes a 5′ phosphate of one or more free nucleotides present in a composition comprising one or more polymerase-nucleotide conjugates.
  • the hydrolysis of the 5’ phosphate prevents the one or more free nucleotides from addition to the nucleic acid during oligonucleotide synthesis.
  • the presence of an unshielded nucleotide in a conjugate reagent can lead to non- termination (i.e., insertion) in oligonucleotide synthesis.
  • an unshielded nucleotide is less likely to inhibit subsequent nucleotide addition after having been added to an oligonucleotide during nucleic acid synthesis.
  • a fraction of nucleotides in a plurality of polymerase- nucleotide conjugates are not shielded by a polymerase.
  • a polymerase-nucleotide conjugate comprises an unshielded nucleotide.
  • a polymerase molecule in a polymerase-nucleotide conjugate does not sterically hinder access of a phosphatase to the 5′ phosphate of a tethered nucleotide.
  • a tethered nucleotide is an unshielded nucleotide.
  • an unshielded nucleotide in a polymerase-nucleotide conjugate is not sterically hindered by a tethered polymerase from a phosphatase capable of removing its 5′ phosphate.
  • removing the 5′ phosphate (e.g., terminal 5’ phosphate) of a nucleotide in a polymerase- nucleotide conjugate prevents the nucleotide from addition to the nucleic acid during nucleic acid synthesis.
  • a phosphatase hydrolyzes the 5′ phosphate (e.g., terminal 5’ phosphate) of an unshielded nucleotide in a polymerase-nucleotide conjugate.
  • more than one 5’ terminal phosphate is removed, for example, wherein a 5’ terminal phosphates are removed serially, i.e., from a first nucleotide, then a second nucleotide, etc.
  • one or more 5’ terminal phosphates may be removed, though in a given nucleotide, a single 5’ terminal phosphate exists and is removed, upon which point a different phosphate becomes the 5’ terminal phosphate of a nucleotide having at least one 5’ terminal phosphate.
  • an unshielded nucleotide is part of an improperly formed conjugate.
  • an improperly formed conjugate comprises a mis-folded polymerase, a polymerase in which a nucleotide is attached in the wrong position, and/or a polymerase in which multiple nucleotides are attached.
  • a nucleotide is free or untethered from a polymerase due to instability or imperfect purification. In some embodiments, a free or untethered nucleotide is an unshielded nucleotide.
  • Lack of shielding of a nucleotide in a polymerase-nucleotide conjugate can occur due to a number of processes during preparation of polymerase-nucleotide conjugates or during the addition reaction itself. Nucleotides that are not attached to a polymerase in a composition comprising a polymerase-nucleotide conjugate are considered unshielded nucleotides.
  • Non-limiting examples of processes that may result in a polymerase-nucleotide conjugate comprising an unshielded nucleotide include: spontaneous cleavage of a linker between a nucleotide and a polymerase (see, e.g., linkers in FIGs. 6A-6D), unfolding of a polymerase (see, e.g., FIG. 6B), a polymerase having a nucleotide attached in the wrong position (see, e.g., FIG. 6A), a polymerase comprising multiple attached nucleotides (i.e., on a single polymerase), or an untethered, free nucleotide (see, e.g., FIG. 6C).
  • spontaneous cleavage of a linker between a nucleotide and a polymerase may occur due to instability, resulting in free nucleotides in the conjugate reagent.
  • a fraction of nucleotides in a plurality of polymerase- nucleotide conjugates are shielded by a polymerase.
  • a non-limiting example of a shielded nucleotide includes a nucleotide that is tethered in the catalytic site of a correctly folded polymerase (see, e.g., exemplary schematic in FIG. 6D).
  • a polymerase-nucleotide conjugate comprises a shielded nucleotide.
  • a polymerase molecule in a polymerase-nucleotide conjugate sterically hinders access of a phosphatase to the 5′ phosphate (e.g., the 5’ terminal phosphate) of a tethered nucleotide.
  • the 5’ phosphate e.g., the 5’ terminal phosphate
  • the 5’ phosphate can be, for example, on an ⁇ -phosphate, ⁇ -phosphate, ⁇ -phosphate, ⁇ -phosphate, ⁇ -phosphate, ⁇ -phosphate, or ⁇ - phosphate.
  • a tethered nucleotide is a shielded nucleotide.
  • a shielded nucleotide in a polymerase-nucleotide conjugate is sterically hindered by a tethered polymerase from a phosphatase capable of removing its 5′ phosphate.
  • a phosphatase is unable to hydrolyze the 5′ phosphate (e.g., 5’ terminal phosphate) of a shielded nucleotide in a polymerase-nucleotide conjugate.
  • Phosphatase [0617] A phosphatase typically uses water to cleave a phosphoric acid monoester into a phosphate ion and an alcohol.
  • a phosphatase enzyme catalyzes the hydrolysis of its substrate.
  • the 5′ phosphate of a nucleotide in a polymerase-nucleotide conjugate is necessary for addition of the nucleotide to an oligonucleotide by a polymerase.
  • removal of the 5′ phosphate of a nucleotide in a polymerase-nucleotide conjugate prevents the nucleotide from addition to the nucleic acid during oligonucleotide synthesis.
  • a phosphatase removes a phosphate moiety from an unshielded nucleotide in a polymerase-nucleotide conjugate.
  • the methods comprise adding a phosphatase capable of hydrolyzing a 5′ phosphate group of an unshielded nucleotide to a polymerase-nucleotide conjugate.
  • any suitable phosphatase, engineered enzyme having phosphatase activity, or a functional fragment thereof for the methods described herein is contemplated by the disclosure.
  • the phosphatase is a nucleotidase.
  • Enzymes having phosphatase activity are included in the enzyme class E.C 3.1.3.-., hydrolases acting on ester bonds, e.g., a phosphoric monoester hydrolase.
  • enzymes having suitable phosphatase activity (such as apyrase) may be found in other enzyme classes.
  • a phosphatase may optionally be capable of hydrolyzing an inorganic phosphate substrate, e.g., pyrophosphate.
  • the phosphatase is immobilized to a solid support.
  • the phosphatase is a fusion protein.
  • the phosphatase comprises a detectable label.
  • the phosphatase is a recombinant polypeptide.
  • the phosphatase is a wild type phosphatase. In some embodiments, the wild type phosphatase is isolated from the organism in which it is natively expressed.
  • Alkaline phosphatases (ALP, ALKP, ALPase, Alk Phos), or basic phosphatases, are plasma membrane-bound glycoproteins that catalyze the hydrolysis of phosphate monoesters and are optimally active at alkaline pH environments. Alkaline phosphatases are homodimeric protein enzymes of 86 kilodaltons. Each monomer contains five cysteine residues, two zinc atoms, and one magnesium atom crucial to its catalytic function. [0623] Non-limiting examples of alkaline phosphatases include: B.
  • Non-alkaline phosphatases may be acid phosphatases.
  • a non-limiting example of a non-alkaline phosphatases is tartrate resistant acid phosphatase.
  • Illustrative amino acid sequences encoding phosphatases for use in the methods described herein are shown, without limitation, in Table 3. Table 3. Exemplary Alkaline Phosphatase Sequences
  • a method of synthesizing a polynucleotide comprises contacting (e.g., incubating) a polymerase-nucleotide conjugate with a nucleic acid, wherein a polymerase of the polymerase-nucleotide conjugate elongates the nucleic acid using its tethered nucleotide.
  • methods of nucleic acid synthesis comprising the step of contacting (e.g., incubating) a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates with a phosphatase.
  • contacting a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates with a phosphatase occurs before or during cyclic extension reactions.
  • the presence of a phosphatase in a stepwise method of nucleic acid synthesis reduces non- terminations and processes that lead to addition of more than one nucleotide per step.
  • use of a conjugate reagent treated with (e.g., incubated with) a phosphatase reduces non-terminations when the conjugate reagent is used in a stepwise method of nucleic acid synthesis as compared to an untreated conjugate reagent.
  • Polymerase-nucleotide conjugates may be stored together with a phosphatase and remain in the system (also during the DNA extension reaction) or a phosphatase may be removed, for a certain incubation period before the conjugates are added to the DNA, and upon initiation of the DNA addition reaction.
  • a phosphatase is incubated with a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates.
  • incubation of a conjugate reagent with a phosphatase is performed before contacting a sample with the conjugate reagent.
  • a phosphatase is removed from a conjugate reagent prior to contacting a sample with the conjugate reagent.
  • incubation of a conjugate reagent with a phosphatase is performed after contacting a sample with the conjugate reagent.
  • concentration of phosphatase in contact or incubated with the polymerase- nucleotide conjugate can be expressed in, for example, a stoichiometric ratio of phosphatase to conjugate fold increase relative to the conjugate concentration, units of activity of phosphatase, molarity, or mg/mL.
  • any suitable stoichiometric ratio of conjugate to phosphatase can be used in the methods described herein.
  • the stoichiometric ratio of conjugate to phosphatase is from about 1:1 to about 1:500.
  • the stoichiometric ratio of conjugate to phosphatase is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:15, about 1:20, about 1:25, about 1:30, about 1:35, about 1:40, about 1:45, about 1:50, about 1:55, about 1:60, about 1:65, about 1:70, about 1:75, about 1:80, about 1:85, about 1:90, about 1:95, about 1:100, about 1:105, about 1:110, about 1:115, about 1:120, about 1:125, about 1:130, about 1:135, about 1:140, about 1:145, about 1:150, about 1:155, about 1:160, about 1:165, about 1:170 about 1:175, about 1:180, about 1:185, about 1:190, about 1:195, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, about 1:425, about 1:
  • any suitable stoichiometric ratio of phosphatase to conjugate can be used in the methods described herein.
  • the stoichiometric ratio of phosphatase to conjugate is from about 1:1 to about 1:500.
  • the stoichiometric ratio of conjugate to phosphatase is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:15, about 1:20, about 1:25, about 1:30, about 1:35, about 1:40, about 1:45, about 1:50, about 1:55, about 1:60, about 1:65, about 1:70, about 1:75, about 1:80, about 1:85, about 1:90, about 1:95, about 1:100, about 1:105, about 1:110, about 1:115, about 1:120, about 1:125, about 1:130, about 1:135, about 1:140, about 1:145, about 1:150, about 1:155, about 1:160, about 1:165, about 1:170 about 1:175, about 1:180, about 1:185, about 1:190, about 1:195, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, about 1:425, about 1:
  • any suitable phosphatase concentration can be used in the methods described herein.
  • the phosphatase concentration is from about 0.01 mg/mL to about 10.5 mg/mL.
  • the phosphatase concentration is about 0.1 mg/mL, about 0.15 mg/mL, about 0.25 mg/mL, about 0.5 mg/mL, about 0.75 mg/mL, about 1 mg/mL, about 1.25 mg/mL, about 1.5 mg/mL, about 1.75 mg/mL, about 2 mg/mL, about 2.25 mg/mL, about 2.5 mg/mL, about 2.75 mg/mL, about 3, about 3.25 mg/mL, about 3.5 mg/mL, about 3.75 mg/mL, about 4 mg/mL, about 4.25 mg/mL, about 4.5 mg/mL, about 4.75 mg/mL, about 5, about 5.25 mg/mL, about 5.5 mg/mL, about 5.75 mg/mL, about 6 mg/mL,
  • any suitable fold increase of phosphatase over conjugate can be used in the methods described herein.
  • the fold increase of phosphatase over conjugate is from about 2-fold to about 500-fold.
  • the fold increase of phosphatase over conjugate is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45- fold, or about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75- fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 105- fold, about 110-fold, about 115-fold, about 120-fold, about 125-fold, about 130-fold, about 135-fold, about 140-fold, about 145-fold, or about 150-fold, about 155-fold, about 160-fold, about 165-fold
  • any suitable fold increase of conjugate over phosphatase can be used in the methods described herein.
  • the fold increase of conjugate over phosphatase is from about 2-fold to about 500-fold.
  • the fold increase of conjugate over phosphatase is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, or about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 105-fold, about 110-fold, about 115-fold, about 120-fold, about 125-fold, about 130- fold, about 135-fold, about 140-fold, about 145-fold, or about 150-fold, about 155-fold, about 160-fold, about 165-fold
  • concentration of phosphatase can be used in the methods described herein.
  • the concentration of phosphatase is from about 0.5 ⁇ M to about 500 ⁇ M.
  • the concentration of phosphatase is about 0.5 ⁇ M, about 1 ⁇ M, about 2 ⁇ M, about 5 ⁇ M, about 10 ⁇ M, about 15 ⁇ M, about 20 ⁇ M, about 25 ⁇ M, about 30 ⁇ M, about 35 ⁇ M, about 40 ⁇ M, about 45 ⁇ M, about 50 ⁇ M, about 55 ⁇ M, about 60 ⁇ M, about 65 ⁇ M, about 70 ⁇ M, about 80 ⁇ M, about 85 ⁇ M, about 90 ⁇ M, about 95 ⁇ M, about 100 ⁇ M, about 105 ⁇ M, about 110 ⁇ M, about 115 ⁇ M, about 120 ⁇ M, about 125 ⁇ M, about 130 ⁇ M, about 135 ⁇ M, about 140 ⁇ M, about 145 ⁇
  • presence of a phosphatase in a stepwise method of nucleic acid synthesis reduces non-terminations and processes that lead to addition of more than one nucleotide per step.
  • Polynucleotides or nucleic acids generated in the methods described herein are said to contain an insertion if a non-termination event has occurred.
  • nucleic acid synthesis in the presence of a phosphatase reduces the rate of non- terminations by about 50% to about 100% compared to nucleic acid synthesis in the absence of a phosphatase.
  • rates of non-terminations are reduced by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% compared to nucleic acid synthesis in the absence of a phosphatase.
  • the total amount of nucleic acid synthesis product with insertions generated in the presence of a phosphatase is less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.1%, less than about 0.05%, or less than about 0.01%.
  • nucleic acid synthesis product generated in the presence of a phosphatase is absent of nucleic acid synthesis product with insertions.
  • methods of synthesizing a polynucleotide comprising a pre-determined sequence comprising contacting a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates with a phosphatase.
  • contacting comprises incubating the conjugate reagent with the phosphatase.
  • the method generates a heterogeneous population of polynucleotide products comprising the pre-determined sequence.
  • the heterogeneous population of polynucleotide products comprising the pre-determined sequence can be referred to as an “end product.”
  • contacting the conjugate reagent comprising a plurality of polymerase-nucleotide conjugates with a phosphatase prevents insertion of nucleotides (i.e., non-terminations), such that these insertions are absent from the pre-determined sequence.
  • an end product comprises nucleic acids, a percentage of which comprise a target sequence and a percentage of which do not comprise a target sequence.
  • an end product comprises less than about 99%, less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.1%, less than about 0.05%, or less than about 0.01% of a polynucleotide comprising a sequence that is not the pre-determined sequence (that is, not the “target” sequence) as compared to a polynucleotide comprising a sequence that is a predetermined (“target”) sequence.
  • the end product is substantially absent of a polynucleotide comprising a sequence that is not the pre-determined (“target”) sequence.
  • Any suitable method known in the art can be used for analyzing the end product.
  • the end product can be assessed by analyzing nucleic acid synthesis products any time following the initiation of an extension reaction (e.g., reaction time course). Analyses can be performed by, for example, capillary electrophoresis (CE) as previously demonstrated (Smith and Nelson. Curr Protoc Nucleic Acid Chem. Chapter 10: Unit 10.9. 2003; Durney et al. Anal Bioanal Chem. 407:6923-6938. 2015).
  • CE capillary electrophoresis
  • CE can separate and report abundance of polynucleotide products with single nucleotide resolution.
  • the relative abundance of each nucleic acid product generated by the methods of nucleic acid synthesis provided herein can be analyzed by CE.
  • the abundance of the starting material i.e., initial polynucleotide or oligonucleotide to which a nucleotide is being incorporated
  • the extension products By comparing the abundance of the starting material (i.e., initial polynucleotide or oligonucleotide to which a nucleotide is being incorporated) and the extension products, it is possible to determine the extent to which the extension reaction is completed. The change over time of the starting material and extended species is indicative of the turnover rate, as described herein. This approach to determine turnover rate has been demonstrated previously (Palluk et al. Nat Biotech. 36(7):645-650. 2018).
  • CE and RP-HPLC may also be used to determine the purity of each species in a nucleic acid synthesis product by determining the area under the curve for peaks in the electropherograms and chromatograms for CE and RP-HPLC, respectively.
  • Any suitable software package suitable for fitting curves to electropherograms and chromatograms and calculating area under the curve (AUC) may be used to determine the abundance of each polynucleotide product in a plurality of nucleotide products.
  • any suitable polynucleotide sequencing method can be used for analysis (e.g., analysis of a sequence of a polynucleotide product, e.g., intermediate product, e.g., end product, etc.).
  • the sequencing method can be long-read sequencing, next generation sequencing, short-read sequencing, shotgun sequencing, sanger sequencing, high throughput sequencing, sequencing by synthesis, sequencing by ligation, sequencing by hybridization, and/or sequencing by mass spectrometry.
  • Sequencing can be suitable for identifying the pre-defined sequence in an end product. Sequencing can be suitable for determining the percentage of sequences in an end product that are not the pre-defined sequence.
  • compositions comprising conjugates comprising nucleotides attached to a polymerase, wherein the purity of nucleotides shielded by a linked polymerase of the composition, as compared to total nucleotides in the composition, is greater than about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, or about 99.9% shielded nucleotides.
  • the purity of nucleotides shielded by a linked polymerase is substantially free of impurities.
  • Low Divalent Cation Concentrations to Improve Stepwise Yield The present disclosure provides insights based on a surprising discovery that rate of extension reactions using polymerase-nucleotide conjugates in polynucleotide synthesis can be increased by reducing the concentration of divalent cation concentrations present in the reactions as compared to standard divalent cation concentrations used in polynucleotide synthesis reactions using a free polymerase and free polynucleotide.
  • the disclosure provides compositions and methods related to the discovery. The details of various embodiments of the compositions and methods are set forth in the disclosure.
  • Nucleic acid synthesis can refer to synthesis, or generation of a product that is a nucleic acid molecule (i.e., a polynucleotide).
  • the methods of nucleic acid synthesis can comprise stepwise synthesis, wherein nucleotides are inserted stepwise into a nucleic acid polymer or polynucleotide.
  • a typical process for stepwise synthesis of a polynucleotide comprises adding nucleotides stepwise to a starter molecule (e.g., an initial oligonucleotide) via the cycled steps of: addition of a polymerase and a nucleotide (e.g., a polymerase-nucleotide conjugate) to an oligonucleotide and covalently incorporating the nucleotide to the 3′ end of the oligonucleotide catalyzed by the polymerase.
  • Successful incorporation of a nucleotide to an oligonucleotide can be referred to as an “extension” or “extension reaction”.
  • the polymerase and nucleotide are linked together (i.e., tethered) to form a conjugate (i.e., a polymerase-nucleotide conjugate).
  • a conjugate i.e., a polymerase-nucleotide conjugate.
  • the tethered nucleotide is covalently incorporated (i.e., is added) into the 3′ end of the oligonucleotide, which is catalyzed by the tethered polymerase.
  • the tethered polymerase can stay tethered to the nucleotide following covalent incorporation.
  • Covalent incorporation of a nucleotide can be referred to as an extension or an addition.
  • the tethered polymerase can be cleaved from the inserted nucleotide to expose the 3′ end of the oligonucleotide. These steps can be repeated to synthesize a desired polynucleotide.
  • the desired polynucleotide can have a pre-determined (i.e., pre-defined or target) sequence.
  • the reaction volume comprises one or more of a buffer, polynucleotide, polymerase-nucleotide conjugate, nucleotide starter molecule, synthesis products, phosphatases, etc.
  • a reaction volume may be prepared comprising only select components (e.g., a buffer and nucleotide starter molecule), and one or more additional components may be added to the volume at one or more subsequent times. In some embodiments, all components for a given synthesis may be added substantially simultaneously.
  • one or more components of a synthesis reaction may be pre-treated (e.g., pretreatment of a polymerase-nucleotide conjugate with a phosphatase) prior to being included in a reaction volume for a nucleic acid synthesis reaction.
  • methods of nucleic acid synthesis disclosed herein are carried out in a reaction buffer composition.
  • the reaction buffer composition is an aqueous solution.
  • the reaction buffer composition comprises a set of components suitable for the stability of the polymerase, nucleotide, polymerase-nucleotide conjugates, starter molecule, nucleic acid molecule products, and any surface or matrix on which the methods disclosed herein are carried out.
  • reaction buffer composition comprises a set of components suitable for carrying out catalytic steps (e.g., polynucleotide polymerization performed by a polymerase) described in the methods of nucleic acid synthesis described herein.
  • catalytic steps e.g., polynucleotide polymerization performed by a polymerase
  • the conditions under which nucleic acid synthesis is carried out can be varied.
  • a synthesis reaction occurs in the presence of one or more divalent cations.
  • the one or more divalent cations is selected from magnesium (Mg 2+ ), calcium (Ca 2+ ), strontium (Sr 2+ ), barium (Ba 2+ ), manganese (Mn 2+ ), cobalt (Co 2+ ), iron (Fe 2+ ), nickel (Ni 2+ ), copper (Cu 2+ ), and/or zinc (Zn 2+ ).
  • the one or more divalent cations is present at a concentration of 2.5 mM or less.
  • the concentration of any given divalent cation present in a nucleic acid synthesis reaction volume is less than about 1 ⁇ M, about 2.5 ⁇ M, about 5 ⁇ M, about 10 ⁇ M, about 15 ⁇ M, about 20 ⁇ M, about 25 ⁇ M, about 30 ⁇ M, about 35 ⁇ M, about 40 ⁇ M, about 45 ⁇ M, about 50 ⁇ M, about 100 ⁇ M, about 150 ⁇ M, about 200 ⁇ M, about 250 ⁇ M, about 300 ⁇ M, about 350 ⁇ M, about 400 ⁇ M, about 450 ⁇ M, about 500 ⁇ M, about 1000 ⁇ M, about 1500 ⁇ M, about 2000 ⁇ M, or about 2500 ⁇ M.
  • the concentration of a divalent cation in a synthesis reaction is 0 ⁇ M or substantially 0 ⁇ M. In some such embodiments, such a divalent cation is considered absence from such a reaction.
  • a concentration of divalent cations present in a synthesis reaction and/or a reaction volume includes a total concentration of divalent cations, which may be comprised of one or more divalent cations.
  • one or more divalent cations are selected from magnesium (Mg 2+ ), calcium (Ca 2+ ), strontium (Sr 2+ ), barium (Ba 2+ ), manganese (Mn 2+ ), cobalt (Co 2+ ), iron (Fe 2+ ), nickel (Ni 2+ ), copper (Cu 2+ ), and zinc (Zn 2+ ).
  • a concentration of divalent cations e.g., in a reaction volume, e.g., of a synthesis reaction
  • one divalent cation e.g., Co 2+ , e.g., Zn 2+
  • a concentration higher e.g., by a certain percent
  • one divalent cation e.g., Co 2+ , e.g., Zn 2+
  • Mg2+ concentration relative to the concentration of one or more other divalent cations
  • one divalent cation e.g., Co 2+ , e.g., Zn 2+
  • a reaction volume may comprise or a synthesis reaction may occur in a total volume wherein the total concentration of divalent cations in the volume comprises at least two cations such as, e.g., Co 2+ and Mg 2+ .
  • a reaction volume includes a total concentration of divalent cations comprised of a single divalent cation (e.g., Co 2+ , Zn 2+ ).
  • the nucleic molecule product i.e., polynucleotide product
  • a “target” or “predetermined” sequence refers to a desired polynucleotide sequence that is intentionally produced by the method of nucleic acid synthesis.
  • the pre-determined sequence can include any number of nucleotides comprising a nucleobase (e.g., adenine, thymine, guanine, cytosine, and/or uracil).
  • the nucleotide is a modified nucleotide (i.e., a nucleotide analog).
  • the nucleobase is a modified nucleobase.
  • the pre-determined sequence contains one or more designated positions which may be a random nucleobase. Inclusion of a position with a random nucleobase can be useful, for example, in introducing a randomized mutation into a polynucleotide product.
  • a nucleic acid molecule product or polynucleotide product generated by the methods described herein can contain a plurality of products.
  • the plurality of products comprises a nucleic acid molecule comprising the target (i.e., pre-determined) sequence.
  • the plurality of products comprises a nucleic acid molecule comprising a sequence that is not the target sequence.
  • the plurality of products comprises a nucleic acid molecule product comprising the target sequence and a nucleic acid molecule product that is not the target sequence.
  • the “purity” of the plurality of products can refer to the ratio of the abundance of nucleic acid molecule products with the target sequence to the abundance of nucleic acid molecule products that do not have the target sequence, for example, taking into account all products and considering which proportion of products does or does not comprise a target sequence. Such purity measurements may be expressed in ways that represent the ratio of one or the other relative to all products, or one relative to the other such as with or without target sequences.
  • the disclosure provides reaction buffer compositions for carrying out a polynucleotide extension reaction (i.e., nucleic acid synthesis reaction buffer).
  • Polymerase enzymes for use in the disclosed methods require a divalent cation cofactor for carrying out the covalent addition of a nucleotide during enzymatic nucleic acid synthesis.
  • the nucleic acid synthesis reaction buffer comprises at least one divalent metal ion. In some embodiments, the concentration of the at least one divalent metal ion is less than about 2500 ⁇ M.
  • the concentration of the at least one divalent cation is less than about 1 ⁇ M, about 2.5 ⁇ M, about 5 ⁇ M, about 10 ⁇ M, about 15 ⁇ M, about 20 ⁇ M, about 25 ⁇ M, about 30 ⁇ M, about 35 ⁇ M, about 40 ⁇ M, about 45 ⁇ M, about 50 ⁇ M, about 100 ⁇ M, about 150 ⁇ M, about 200 ⁇ M, about 250 ⁇ M, about 300 ⁇ M, about 350 ⁇ M, about 400 ⁇ M, about 450 ⁇ M, about 500 ⁇ M, about 1000 ⁇ M, about 1500 ⁇ M, about 2000 ⁇ M, or about 2500 ⁇ M.
  • the concentration of a given divalent cation in a nucleic acid synthesis reaction is 0 ⁇ M or substantially 0 ⁇ M. In some such embodiments, such a divalent cation is considered absent from such a reaction.
  • the at least one divalent cation is selected from magnesium (Mg 2+ ), calcium (Ca 2+ ), strontium (Sr 2+ ), barium (Ba 2+ ), manganese (Mn 2+ ), cobalt (Co 2+ ), iron (Fe 2+ ), nickel (Ni 2+ ), copper (Cu 2+ ), and zinc (Zn 2+ ), or a combination thereof.
  • the at least one divalent cation is Mg 2+ . In some embodiments, the at least one divalent cation is Ca 2+ . In some embodiments, the at least one divalent cation is Sr 2+ . In some embodiments, the at least one divalent cation is Ba 2+ . In some embodiments, the at least one divalent cation is Mn 2+ . In some embodiments, the at least one divalent cation is Co 2+ . In some embodiments, the at least one divalent cation is Fe 2+ . In some embodiments, the at least one divalent cation is Ni 2+ . In some embodiments, the at least one divalent cation is Cu 2+ .
  • the at least one divalent cation is Zn 2+ . [0661] In some embodiments, the at least one divalent cation is not Mg 2+ . In some embodiments, the at least one divalent cation is not Ca 2+ . In some embodiments, the at least one divalent cation is not Sr 2+ . In some embodiments, the at least one divalent cation is not Ba 2+ . In some embodiments, the at least one divalent cation is not Mn 2+ . In some embodiments, the at least one divalent cation is not Co 2+ . In some embodiments, the at least one divalent cation is not Fe 2+ .
  • the at least one divalent cation is not Ni 2+ . In some embodiments, the at least one divalent cation is not Cu 2+ . In some embodiments, the at least one divalent cation is not Zn 2+ .
  • a nucleic acid synthesis reaction comprising at least one divalent cation that is not Mg 2+ proceeds at a faster rate than an identical reaction that comprises Mg 2+ (either alone or in the presence of one or more other divalent cations).
  • a nucleic acid synthesis reaction occurs under conditions in which magnesium is not present or is present in a concentration lower than a concentration of at least one other divalent cation.
  • the nucleic acid synthesis reaction buffer comprises a pH buffering component.
  • the buffering component is used at a concentration from 1 mM to IM in the nucleic acid synthesis reaction buffer. In some embodiments, the buffering component is at a concentration of about 10 mM to about 100 mM. In some embodiments, the buffering component is at a concentration of about 100 to about 200 mM. In some embodiments, the buffering component is at a concentration of about 50 mM to about 100 mM. In some embodiments, the buffering component is at a concentration of about 10 mM to about 50 mM. In some embodiments, the buffering component is about a concentration of about 20 mM.
  • Illustrative buffering components includes, without limitation, Tris (tris(hydroxymethyl)aminomethane), Tricine, bicine, Bis-Tris, CAPS, EPPS, HEPES (4-(2- hydroxyethyl)-l -piperazineethanesulfonic acid), MES, MOPS, PIPES, TAPS and TES.
  • the nucleic acid synthesis reaction buffer comprises Tris.
  • the nucleic acid synthesis reaction buffer comprises HEPES.
  • the nucleic acid synthesis reaction buffer has a pH from about pH 6.0 to about pH 8.5. In some embodiments, the pH is about pH 6.0 to about pH 8.5. In some embodiments, the pH is about pH 6.5 to about pH 8.0. In some embodiments, the pH is about pH 7 to about pH 7.5. In some embodiments, the pH is about pH 7.5 to about pH 8.0. In some embodiments, the pH is about pH 8.
  • the nucleic acid synthesis reaction buffer comprises a monovalent cation.
  • the monovalent cation may be a salt.
  • the monovalent cation is selected from sodium, potassium, lithium, rubidium, cesium, ammonium or any combination thereof.
  • the monovalent cation is at a concentration of about 100 to about 200 mM
  • the nucleic acid synthesis reaction buffer comprises a detergent, surfactant, or nonionic surfactant.
  • the detergent, surfactant, or nonionic surfactant is selected from TRITON X-100®, Nonidet P-40 (NP-40), Tween 20, P20, and Brij 35, or any combination thereof.
  • the nucleic acid synthesis reaction buffer comprises one or more stabilizing agents.
  • the one or more stabilizing agents is bovine serum albumin and/or glycerol.
  • the nucleic acid synthesis reaction buffer comprises one or more reducing agents.
  • the reducing agent is selected from dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP), and P-mercaptoethanol.
  • reagent components are mixed at working concentrations to form a solution suitable for immediate use with or without dilution or addition of further reagents.
  • the water used in the formulations of the present disclosure can be distilled, deionized and sterile filtered (through a 0.1-0.2 micrometer filter), and is free of contamination by DNase and RNase enzymes.
  • Such water is available commercially, for example from Sigma Chemical Company (Saint Louis, Mo.), or may be made as needed according to methods well known to those skilled in the art.
  • a method of nucleic acid synthesis comprising providing a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates, wherein said conjugates comprise a nucleotide or a modified nucleotide covalently linked to a polymerase via a linker, and contacting a sample comprising a polynucleotide with said conjugate reagent, wherein said polymerase of the conjugate catalyzes an extension reaction comprising the covalent addition of a shielded nucleotide of the conjugate onto the 3' hydroxyl of said polynucleotide in the nucleic acid synthesis reaction buffer disclosed herein.
  • a method of nucleic acid synthesis comprises contacting a polynucleotide with a polymerase and a nucleotide, wherein the effective concentration of the nucleotide relative to the polymerase has been increased artificially.
  • the method is carried out in the nucleic acid synthesis reaction buffer disclosed herein.
  • an effective concentration of the nucleotide relative to the polymerase can be increased artificially by, for instance, engineering a polymerase to have greater affinity for the nucleotide to be incorporated by the polymerase in an extension reaction or tethering the nucleotide to the polymerase.
  • the extension reaction has a faster turnover rate than an extension reaction performed in the presence of at least one divalent cation at a concentration greater than about 1 ⁇ M, about 2.5 ⁇ M, about 5 ⁇ M, about 10 ⁇ M, about 15 ⁇ M, about 20 ⁇ M, about 25 ⁇ M, about 30 ⁇ M, about 35 ⁇ M, about 40 ⁇ M, about 45 ⁇ M, about 50 pM, about 100 ⁇ M, about 150 ⁇ M, about 200 ⁇ M, about 250 ⁇ M, about 300 ⁇ M, about 350 pM, about 400 ⁇ M, about 450 ⁇ M, about 500 ⁇ M, about 1000 ⁇ M, about 1500 ⁇ M, about 2000 ⁇ M, about 2500 pM.
  • the faster turnover rate is between about 1 second faster and about 60 seconds faster. In some embodiments, the faster turnover rate is about 1 second faster, about 2 seconds faster, about 3 seconds faster, about 4 seconds faster, about 5 seconds faster, about 6 seconds faster, about 7 seconds faster, about 8 seconds faster, about 9 seconds faster, about 10 seconds faster, about 15 seconds faster, about 20 seconds faster, about 25 seconds faster, about 30 seconds faster, about 35 seconds faster, about 40 seconds faster, about 45 seconds faster, about 50 seconds faster, about 55 seconds faster, or about 60 seconds faster.
  • the faster turnover rate is between about 60 seconds faster and about 300 seconds faster. In some embodiments, the faster turnover rate is about 70 seconds faster. In some embodiments, the faster turnover rate is about 80 seconds faster. In some embodiments, the faster turnover rate is about 90 seconds faster. In some embodiments, the faster turnover rate is about 100 seconds faster. In some embodiments, the faster turnover rate is about 110 seconds faster. In some embodiments, the faster turnover rate is about 120 seconds faster. In some embodiments, the faster turnover rate is about 130 seconds faster. In some embodiments, the faster turnover rate is about 140 seconds faster. In some embodiments, the faster turnover rate is about 150 seconds faster. In some embodiments, the faster turnover rate is about 160 seconds faster. In some embodiments, the faster turnover rate is about 170 seconds faster.
  • the faster turnover rate is about 180 seconds faster. In some embodiments, the faster turnover rate is about 190 seconds faster. In some embodiments, the faster turnover rate is about 200 seconds faster. In some embodiments, the faster turnover rate is about 210 seconds faster. In some embodiments, the faster turnover rate is about 220 seconds faster. In some embodiments, the faster turnover rate is about 230 seconds faster. In some embodiments, the faster turnover rate is about 240 seconds faster. In some embodiments, the faster turnover rate is about 250 seconds faster. In some embodiments, the faster turnover rate is about 260 seconds faster. In some embodiments, the faster turnover rate is about 270 seconds faster. In some embodiments, the faster turnover rate is about 280 seconds faster. In some embodiments, the faster turnover rate is about 290 seconds faster. In some embodiments, the faster turnover rate is about 300 seconds faster.
  • the methods of nucleic acid synthesis provided herein comprise a faster turnover rate for an extension reaction comprising the nucleic acid synthesis reaction buffer described herein.
  • the turnover rate refers to the time required to extend an oligonucleotide by at least one nucleotide.
  • any suitable method known in the art can be used for determining the turnover rate.
  • the turnover rate can be assessed by analyzing nucleic acid synthesis products over time following the initiation of an extension reaction (i.e., reaction time course). Analyses can be performed by, for example, capillary electrophoresis (CE) as previously demonstrated (Smith and Nelson. Curr Protoc Nucleic Acid Chem. Chapter 10: Unit 10.9. 2003; Dumey et al. Anal Bioanal Chem. 407:6923-6938. 2015).
  • CE can separate and report abundance of polynucleotide products with single nucleotide resolution. The relative abundance of each nucleic acid product generated by the methods of nucleic acid synthesis provided herein can be analyzed by CE.
  • the abundance of the starting material i.e., initial polynucleotide or oligonucleotide to which a nucleotide is being incorporated
  • the expected polynucleotide product By comparing the abundance of the starting material (i.e., initial polynucleotide or oligonucleotide to which a nucleotide is being incorporated) and the expected polynucleotide product, it is possible to determine the extent to which the extension reaction is completed. The change over time of the starting material and extended species is indicative of the turnover rate, as described herein. This approach to determine turnover rate has been demonstrated previously (Palluk et al. Nat Biotech. 36(7):645-650. 2018). Alternatively, analysis of nucleic acid synthesis products can be performed using reverse- phase high-performance liquid chromatography (RP-HPLC) as described previously (Jensen and Davis. Biochemistry. 57(12): 1821-1832. 2018).
  • RP-HPLC reverse- phase high-
  • CE and RP-HPLC may also be used to determine the purity of each species in a nucleic acid synthesis product by determining the area under the curve for peaks in the electropherograms and chromatograms for CE and RP-HPLC, respectively.
  • Any suitable software package suitable for fitting curves to electropherograms and chromatograms and calculating area under the curve (AUC) may be used to determine the abundance of each polynucleotide product in a plurality of nucleotide products.
  • the steps are performed by dipping a reaction surface comprising a bound synthesis initiator (which may also include previously added monomers) into a contained solution comprising the desired reagents.
  • the method is amenable to multiplexed polymer synthesis, such that a plurality of elements having an end with the reaction surface and bound synthesis initiator may be used and simultaneously dipped into a plurality of contained liquid reagents (such as in wells or droplets on a surface) aligned with the reaction surfaces.
  • the contained liquid reagent comprise a polymer extension solution with a monomer of a specific identity and enzyme capable of adding the monomer to the synthesis initiator.
  • synthesis of a polymer comprises adding blocked monomers stepwise to a synthesis initiator (e.g., an initial oligonucleotide) via the cycled steps of: addition of monomer comprising a blocking group (i.e., a blocked monomer) to a synthesis initiator or extended polymer comprising previously added monomers, binding of the monomer to the end of the synthesis initiator or extended polymer catalyzed by the enzyme, and removal of the blocking group from the monomer to allow addition of a subsequent monomer to the extended polymer.
  • a blocking group i.e., a blocked monomer
  • a “blocking group” bound to the monomer is a group capable of preventing addition of another monomer once the monomer has been added to the synthesis initiator or extended polymer. After addition of the desired blocked monomer and removal of excess monomer during an extension cycle, the extended polymer is immersed in a monomer deblocking solution capable of removing the blocking group from the monomer.
  • the blocking group can be the enzyme that catalyzes addition of the monomer to the synthesis initiator or extended polymer, wherein the enzyme is linked to the monomer (i.e., a monomer-enzyme conjugate).
  • the enzyme can sterically hinder addition of a subsequent monomer after addition of the blocked monomer to the polymer.
  • a monomer deblocking solution that removes the enzyme from the monomer can then be used, such as a linker cleavage solution.
  • Both the monomer addition and blocking group removal steps may be quenched by immersing the extended polymer in an appropriate reaction quenching solution, such as EDTA. In addition, washing steps may be used between steps by immersing the extended polymer in a wash buffer.
  • a system for performing steps in a method of polynucleotide synthesis to generate polynucleotides of desired length and sequence according to the embodiments described herein.
  • the steps are performed by dipping a reaction surface comprising a bound synthesis initiator (which may also include previously added nucleotides) into a contained solution comprising the desired reagents.
  • the method is amenable to multiplexed polynucleotide synthesis, such that a plurality of elements having an end with the reaction surface and bound synthesis initiator may be used and simultaneously dipped into a plurality of contained liquid reagents (such as in wells or droplets on a surface) aligned with the reaction surfaces.
  • the contained liquid reagent comprise a polymer extension solution with a nucleotide of a specific identity and polymerase capable of adding the nucleotide to the synthesis initiator.
  • synthesis of a polynucleotide comprises adding blocked nucleotides stepwise to an oligonucleotide bound to the reaction surface on the element via the cycled steps of: addition of nucleotide comprising a blocking group (i.e., a blocked nucleotide) to a synthesis initiator or extended polynucleotide comprising previously added nucleotides, binding of the nucleotide to the end of the synthesis initiator or extended polynucleotide catalyzed by the polymerase, and removal of the blocking group from the nucleotide to allow addition of a subsequent nucleotide to the extended polynucleotide.
  • the blocking group bound to the nucleotide is a group capable of preventing addition of another nucleotide once the nucleotide has been added to the synthesis initiator or extended polynucleotide.
  • the extended polynucleotide is immersed in a nucleotide deblocking solution capable of removing the blocking group from the nucleotide.
  • the blocking group is the polymerase that catalyzes addition of the nucleotide to the surface-bound polynucleotide, wherein the polymerase is linked to the nucleotide (z.e., a nucleotide-polymerase conjugate).
  • the polymerase can sterically hinder addition of a subsequent nucleotide after addition of the blocked nucleotide to the polynucleotide.
  • a monomer deblocking solution that removes the polymerase from the nucleotide can then be used to remove the blocking group, such as a linker cleavage solution.
  • the blocking group is a reversible terminator bound to the nucleotide.
  • a monomer deblocking solution that removes the reversible terminator from the nucleotide can then be used to remove the blocking group, such as a linker cleavage solution.
  • both a reversible terminator and a polymerase bound to the nucleotide may be used.
  • Both the nucleotide addition and blocking group removal steps may be quenched by immersing the extended polynucleotide in an appropriate reaction quenching solution, such as EDTA.
  • washing steps may be used between steps by immersing the extended polynucleotide in a wash buffer.
  • Exemplary embodiments may provide methods and systems for polymer synthesis, such as de novo enzymatic polynucleotide synthesis.
  • synthesis surfaces on elements may have a base molecule bound to the synthesis surfaces.
  • the synthesis surfaces may be placed in contact with liquid reagent volumes, such as a polymer extension solution comprising a monomer and an enzyme capable of catalyzing addition of the monomer to a synthesis initiator or polymer, to synthesize polymers.
  • the system may include an array of elements that have the functionalized surfaces at least on their distal portions, and the elements may be moved by one or more actuators to be contacted with (i.e., at least partially immersed) into a liquid reagent in a resolved loci on a surface, such as a plate of wells or a plate with a patterned surface holding a polymer extension solution, to perform the synthesis.
  • a surface such as a plate of wells or a plate with a patterned surface holding a polymer extension solution
  • Resolved loci are fluidically disconnected from each other and capable of containing a liquid reagent on a surface separate from other liquid reagents, e.g., a substrate or plate can have a first resolved locus capable of containing a first polymer extension solution (such as a contained droplet or well) for a first oligonucleotide and a second resolved locus capable of containing a second polymer extension solution (such as a contained droplet or well) for a second oligonucleotide, the first and second polymer extension solutions being fluidically isolated from each other (and other polymer extension solutions) on the surface or plate.
  • a first polymer extension solution such as a contained droplet or well
  • second resolved locus capable of containing a second polymer extension solution (such as a contained droplet or well) for a second oligonucleotide
  • a plate comprising resolved loci is a droplet microarray comprising a substrate having a hydrophobic -hydrophilic patterned surface on which a plurality of resolved loci correspond to hydrophilic locations each of which is capable of hosting a liquid reagent.
  • Preparation of substrates with discrete resolved loci for containing liquid reagent can be accomplished by known methods. For example, such methods can involve the creation of hydrophilic reaction sites by first applying a protectant, or resist, over selected areas over the surface of a substrate, such as a silicon oxide, or like material.
  • the unprotected areas are then coated with a hydrophobic agent to yield an unreactive surface.
  • a hydrophobic coating can be created by chemical vapor deposition of (tridecafluorotetrahydrooctyl)-triethoxysilane onto the exposed oxide surrounding the protected circles.
  • the protectant, or resist is removed exposing the well regions of the array for further modification and nucleoside synthesis using the high surface tension solvents described herein and procedures known in the art such as those described by Maskos & Southern, Nucl. Acids Res. 20:1679-1684 (1992).
  • a glass plate substrate can be coated with hydrophobic material, such as 3-(l,l- dihydroperfluoroctyloxy)propyltriethoxysilane, which is ablated at desired loci to expose the underlying silicon dioxide glass.
  • hydrophobic material such as 3-(l,l- dihydroperfluoroctyloxy)propyltriethoxysilane, which is ablated at desired loci to expose the underlying silicon dioxide glass.
  • the substrate is then coated with glycidyloxypropyl trimethoxysilane, which reacts only with the glass, and which is subsequently “treated” with hexaethylene glycol and sulfuric acid to form a hydroxyl group-bearing linker upon which chemical species can be synthesized (Brennan, U.S. Pat. No. 5,474,796).
  • Arrays produced in such a manner can localize small volumes of solvent within the reaction site by virtue of surface tension effects (Lopez et ah
  • the number of elements may be matched with the number and positioning of wells on a plate or locations on a patterned surface plate holding a polymer extension solution or other liquid reagent volume.
  • the systems of the exemplary embodiments may also perform operations, such as hybridization of the functionalized surfaces, heating or cooling of the liquid reagent volumes, agitation of the liquid reagent volumes and washing of the dipping elements (i.e., synthesis elements) and/or plates in order to properly realize the synthesis.
  • the exemplary embodiments may enable reduced polymer synthesis times by reducing the time required for each cycle of synthesis.
  • the approach of these exemplary embodiments separates the loading operations of loading wells on a plate or resolved loci on a patterned surface with the liquid reagent volumes from the synthesis operations involving contacting synthesis surfaces with the liquid reagent volumes. This separation of the fluidic steps from the synthesis steps helps to reduce cycle time as the fluidic steps are performed beforehand and need not be performed as part of the synthesis steps.
  • the liquid reagent volumes may be pre-mixed in the wells or the resolved loci on the patterned surface. The synthesis may be quicker than conventional polymer synthesis systems.
  • the delivery of reagent to the polymer by dipping the polymer into a reagent will also mitigate errors in DNA synthesis caused by conventional methods of reagent delivery to the reaction site, such as bubble formation or incomplete reagent delivery.
  • any issues with reagent delivery could result in inhibition of a synthesis or deblocking reaction.
  • re-dipping the synthesis surface comprising the attached polymer or synthesis initiator can lead to increased robustness as the reaction (e.g., monomer addition or deblocking) is initiated more than once, mitigating any issues that may arise during a single dip or a single reagent delivery.
  • a polymer may be dipped (e.g., immersing the polymer / synthesis surface into a liquid reagent compartmentalized in at a resolved locus) and re- dipped into the same liquid reagent (e.g., a polymer extension solution) multiple times to agitate the solution and/or to reduce the impact liquid reagent delivery problems, such as bubble formation or incomplete reagent delivery.
  • a liquid reagent e.g., a polymer extension solution
  • a desired reaction from one instance of dipping the polymer into the liquid reagent may be inhibited as described above, repeated dipping into the liquid reagent allows for additional attempts to perform the reaction in a different configuration, e.g., a bubble may be moved so that it does not inhibit the reaction in at least some of the dips.
  • the exemplary embodiments are applicable to polymer synthesis and are particularly applicable to enzymatic polynucleotide synthesis as described below.
  • the discussion below generally focuses on polymer synthesis but, in some instances, focuses on polynucleotide synthesis.
  • FIG. 7 depicts a block diagram of components of an illustrative synthesis system 100 for performing polymer synthesis, such as enzymatic polynucleotide synthesis, in exemplary embodiments. Illustrative configurations of these components are discussed below.
  • the synthesis system 100 may include an element array 102.
  • the element array 102 may include a number of elements organized in an array. As is explained below, each element may include a synthesis surface or surfaces on at least a portion of the element to which a base molecule is bound or will be bound.
  • the synthesis surface comprises a functionalized surface capable of binding to the base molecule.
  • a polynucleotide modified with a disulfide group can be covalently bound to a surface that has been modified with a thiol group (-SH).
  • Other functional groups that are useful for binding oligonucleotides include amino groups (-NH2) and carboxyl groups (-COOH).
  • surfaces such as glass or silicon oxide can be functionalized with silanes such as aminopropyltrimethoxy silane (APTMS) and these can be used to covalently bind amine- modified oligonucleotides.
  • ATMS aminopropyltrimethoxy silane
  • gold, silver, and titanium surfaces can be functionalized with thiol groups and can be used for immobilizing thiolated oligonucleotides to form a “self-assembled monolayers (SAM)”.
  • SAM self-assembled monolayers
  • a functionalized surface is chemically or naturally charged like glass or silicon, where oligonucleotides can be adsorbed via electrostatic interactions.
  • surfaces such as glass or silicon oxide can be functionalized with silanes such as aminopropyltrimethoxy silane (APTMS) and these can be used to covalently bind amine-modified oligonucleotides.
  • silanes such as aminopropyltrimethoxy silane (APTMS) and these can be used to covalently bind amine-modified oligonucleotides.
  • a base molecule is a synthesis initiator comprising a reactive group for coupling to a monomer in a cycled polymer synthesis reaction.
  • a polynucleotide sequence bound to a synthesis surface and having a free 3' hydroxyl at its end may be a synthesis initiator.
  • a synthesis initiator comprises a non- nucleic acid compound having a free hydroxyl to which a TdT may couple a nucleotide, e.g., as described in U.S. patent publications US2019/0078065 and US2019/0078126.
  • a base molecule is a compound bound to the reaction surface and is capable of binding to the reaction initiator.
  • a reaction initiator oligonucleotide having a sufficiently complementary sequence to hybridize to a base molecule oligonucleotide bound to the surface at its 3' end may be used.
  • the synthesis reaction does not occur off of the covalently bound base molecule but occurs at the end of the hybridized oligonucleotide.
  • an additional step of hybridization of the reaction initiator to the base molecule bound to the reaction surface may be bound.
  • the system may only comprise a base molecule bound to the surface without a bound reaction initiator before use in the processes described herein.
  • a system comprising a base molecule bound to the reaction surface of an element can include a reaction initiator (when the base molecule is the reaction initiator) or is primed to bind to a reaction initiator (i.e., comprises a base molecule capable of binding to the reaction initiator).
  • the number of elements in an array and the arrangements of the elements may match that of a group of wells in a plate of wells or of other resolved loci, such as a group of hydrophilic locations in a patterned surface plate that includes a pattern of hydrophilic locations and hydrophobic locations.
  • the elements of the element array 102 may be held by an element array holder 104.
  • substrates may be employed for creating synthesis surfaces or elements for enzymatic synthesis of polynucleotides.
  • Substrates may be a rigid material including, without limitation, glass; fused silica; silicon such as silicon dioxide or silicon nitride; metals such as gold or platinum; plastics such as polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and any combination thereof.
  • a rigid surface can be fabricated from a material selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane (PDMS), and glass.
  • Substrates may also comprise flexible materials, which is capable of being bent, folded, or similarly manipulated without breakage.
  • exemplary flexible materials include, without limitation, nylon (unmodified nylon, modified nylon, clear nylon), nitrocellulose, polypropylene, polycarbonate, polyethylene, polyurethane, polystyrene, acetal, acrylic, acrylonitrile, butadiene styrene (ABS), polyester films such as polyethylene terephthalate, polymethyl methacrylate or other acrylics, polyvinyl chloride or other vinyl resin, transparent PVC foil, transparent foil for printers, Poly (methyl methacrylate) (PMMA), methacrylate copolymers, styrenic polymers, high refractive index polymers, fluorine-containing polymers, polyethersulfone, polyimides containing an alicyclic structure, rubber, fabric, metal foils, and any combination thereof.
  • the element array holder 104 may be actuatable by actuator(s) 106, which may actuate one or more actuatable components 109, such as a robotic arm, that may hold or contain the element array holder 104.
  • the one or more actuator(s) 106 may include driver(s)
  • magnetic elements and electrical elements may serve as the driver(s) 108.
  • the driver(s) 108 may drive motion of the one or more actuatable components 109 to move up or down, move laterally and/or even rotate.
  • the driver 108 may cause linear actuation in the X and Y dimensions of a plane to position the elements of the element array 102 to be in alignment above resolved loci of the plate (e.g., the wells of a plate or hydrophilic locations on a patterned surface plate) 128 and to move the elements into contact with the liquid reagent volumes in the resolved loci of the plate 128 as part of the polymer synthesis.
  • multiple drivers 108 may be provided.
  • the actuation by the one or more actuator(s) 106 may be under the control of a processor 110 that executes computer programming instructions of control application 112 stored in a storage 114.
  • the control application 112 may include computer programming instructions for controlling operations of the synthesis system 100.
  • the control application 112 may invoke operations and direct activities of the components of the synthesis system 100 described herein 100.
  • the processor 110 may be a central processing unit (CPU), a graphics processing unit (GPU), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA), or other type of processor. In some exemplary embodiments, more than one processor may be provided.
  • the storage 114 may be a non- transitory computer-readable storage medium, such as one or more of random access memory (RAM), read only memory (ROM), solid state memory, magnetic disk storage, optical disk storage or the like.
  • the synthesis system 100 may include a display 116 for displaying, graphical textual and video content.
  • the display 116 may display a user interface (UI) 118 under the control of the control application 112.
  • the UI 118 may display useful information to a user of the synthesis system 100 and may be used to enter commands or invoke operations in the synthesis system.
  • a user may cause actuation of the one or more actuators 106 by entering commands via input devices 120, such as a mouse, a keyboard, a thumbpad or the like.
  • the display 116 may be a touchscreen that is capable of receiving input by the user touching the screen of the display 116.
  • the input devices 120 may also include knobs, levers and buttons that are used to control operation of the synthesis system 100.
  • the processor 110, storage 114, the display 116 and the input devices 120 may be part of a computer system, such as a workstation, personal computer, laptop computer, tablet computer, smartphone, or other computing device, that is coupled to the other components of the synthesis system 100.
  • the coupling may be a hardwired connection or a network connection, such as via a wired or wireless network.
  • these components 110, 114, 116 and 120 may be integrated as part of the synthesis system 100, such as in a common housing or in one of multiple housings of the synthesis system 100.
  • the synthesis system 100 may also include a plate washer 122 for washing reaction plates.
  • the washing reaction plates can include a uniform bath for immersing multiple reaction surfaces into.
  • a wash solution can be contained in wells or patterned surfaces 128 that are used in the synthesis system 100.
  • the synthesis system 100 may include a washing fountain 124.
  • Dispenser(s) 126 may be provided for dispensing solution to the resolved loci on plates or patterned surfaces 128.
  • the dispenser(s) 126 may have, for example, fluidic connections with a reagent holder 127A containing a wash solution, a reagent container 127B containing a quenching solution for quenching a reaction, a reagent container 127C containing a deblocking solution and a reagent container 127D, containing a reagent solution, such as a polymer extension solution.
  • a thermal controller 130 may be provided for heating and/or cooling the reaction plates or patterned surfaces 128 or the liquid reagent volumes held therein. This may ensure that the reagents involved in the synthesis are at a suitable temperature for reactions that occur during the polymer synthesis.
  • a shaker 132 may be provided for shaking the reaction plates or patterned surfaces 128 as needed to properly complete the polymer synthesis.
  • FIG. 8 depicts a first illustrative configuration 200 of the synthesis system.
  • an element array 202 is held in an element array holder 204.
  • the element array holder 204 is secured in an arm 205 that is actuatable by an actuator (not shown), like an electric motor.
  • the element array holder 204 may be moved up and down (i.e., along the Z axis) relative to the plane of the top surface of the reaction plate 206, where at least some of the resolved loci hold a liquid reagent volume.
  • the element array holder 204 is positioned on a platform 208.
  • the platform 208 is actuatable in the X direction.
  • the platform 208 may be actuated to slide along the rail 210 to be positioned under the element array 202 so that at least some of the elements are in alignment with at least some of the resolved loci (e.g., wells) of the plate 206.
  • the elements may be placed in contact with the liquid reagent volumes in the resolved loci as will be described below as part of the synthesis. Subsequently, the platform 208 may be actuated to slide on the rail 210 back to the position shown in FIG. 8.
  • Plates 212 are positioned on a platform 218.
  • the platform 218 may slide along rail 214 in the X direction and/or rail 216 in the Y direction.
  • the plates 212 may include a plate where at least some of the resolved loci contain quenching solution, and a plate where at least some of the resolved loci contain cleaving solution.
  • One or more of the reaction plates 212 may contain wells holding a wash solution.
  • the platform 218 may be actuated by one or more of the actuators to move individual one of the plates 212 under the element array 202 so that elements may be placed in contact with contents in at least some of the resolved loci of the individual plates. This facilitates performing quenching, cleaving, and washing steps as described below.
  • FIG. 9 depicts a second illustrative configuration 300 of the synthesis system.
  • plates 301 ride on levitating carriers 304.
  • the levitating carriers 304 are levitated and propelled over a platform 302 by magnets and electrical components found in magnetic levitation systems.
  • the levitating carriers are controlled using two sets of magnets: one set that provides the lifting force and another set that provides the guidance and stability.
  • superconducting magnets are used.
  • the levitating carrier can be made of a magnetic material, such as iron, and has a magnetization that is parallel to the direction of the lifting force.
  • the direction of the magnetic field can be changed. This can be done by adjusting the current in the superconducting magnets or by using additional magnets that can generate a secondary magnetic field. This change in the direction of the magnetic field will cause the plate to move, following the direction of the magnetic field.
  • magnetic levitation is performed using permanent magnets. In this case, a combination of repulsive and attractive forces between the magnets are used to create the levitation. To move the levitated plate, the magnetic field and position of the permanent magnets are adjusted.
  • the levitating carriers 304 are actuated under computer programming control to move between stations during the synthesis process.
  • the levitating carriers 304 may be actuated to the dispensing subsystem 306.
  • the dispensing subsystem station 306 contains dispensers for dispensing liquid reagent volumes, such as a polymer extension solution, in at least some of the wells of the plates 301.
  • the bath fill, drain subsystem station 308 may contain dispensers for dispensing quenching solution, cleaving solution and wash solution to wells of reaction plates 301.
  • the levitating carriers 304 may also be actuated to be positioned at a thermal subsystem station 312.
  • the thermal subsystem station 312 may contain heating elements and/or cooling elements that may be placed in contact with the reaction plates 301 or the liquid reagent volumes of at least some of the resolved loci of the plates 301 to adjust the temperature of the liquid reagent volumes.
  • the levitating carriers 304 may be actuated to be positioned at the dipper subsystem station 310.
  • the dipper subsystem station 310 may include an element array that is actuatable up and down relative to the planar upper face of the platform 302. The actuation of the element array may bring at least some of the elements of the element array into contact with liquid reagent volumes in at least some of the wells of reaction plates 301 as part of the synthesis process.
  • the element array may also be actuated to bring at least some of the elements into contact with a quenching solution, a deblocking solution and/or a wash solution in wells of the plates 301 as part of the synthesis process.
  • FIG. 10 depicts an illustrative element 400 and an illustrative well that are suitable for the exemplary embodiments.
  • the element 400 is a cylindrical rod.
  • the element 400 may be made of suitable materials, such as stainless steel, glass, plastic or the like.
  • the element 400 may have one or more synthesis surfaces 404 on which a base molecule, like a polynucleotide, is bound.
  • the distal end of the element 400 is where the synthesis surface 404 is located.
  • the synthesis surface may extend about the circumference of the cylinder of the element 400.
  • the remainder of the length of the element extending from the synthesis surface 404 to the proximal end of the element 400 may be covered with a hydrophobic coating, such as polytetrafluoroethylene (PTFE), perylene, polyimide or another coating.
  • a hydrophobic coating such as polytetrafluoroethylene (PTFE), perylene, polyimide or another coating.
  • PTFE polytetrafluoroethylene
  • the hydrophobic coating 406 limits the amount of unintended functionalization and extension beyond the functionalized surface.
  • a well 402 is formed in the plate 408.
  • the well 402 holds a liquid reagent volume, such as a polymer extension solution (e.g., a polymerase-nucleotide conjugate solution), 410 that has been dispensed to the well 402.
  • a polymer extension solution e.g., a polymerase-nucleotide conjugate solution
  • the shape and size of the well and the dipping element 400 are chosen so that the dipping element may be actuated downward into the well 402 to contact the polymer extension solution 410 as part of the synthesis. Specifically, the actuation at least partially immerses the distal portion of the functionalized surface 404 into the polymer extension solution 410.
  • the distal portion of the functionalized surface 404 may remain in the polymer extension solution 410 for a predetermined period of time (e.g., 30 to 90 seconds) and then is removed by actuating the dipping element 400 out of the well 402.
  • a predetermined period of time e.g. 30 to 90 seconds
  • the downward and upward actuation are indicated in FIG. 10 by the arrow 412.
  • the elements may assume a number of different configurations.
  • the examples depicted thus far have been cylindrical rods with a circular cross-sections 502, such as shown in FIG. 11.
  • the cross-section of an element need not be circular. Instead, the cross-section of an element may be, for example, oval 504 or triangular 506. Further, the cross-section of an element may be square 508 or rectangular 510.
  • These cross-section shapes shown in FIG. 11 are intended to be illustrative and not limiting. The elements may have other cross- sectional shapes that are not shown.
  • FIG. 12 depicts a longitudinal side view of various alternate element designs.
  • An element may be a rectangular plate 602 with a rectangular face of small thickness.
  • An element may be a small a straight filament 604.
  • the element may be an oval plate 606 with a wide oval face and a small thickness.
  • an element may be a diamond- shaped plate 608 with a wide face and a small thickness.
  • the elements may be held by the element array so as to “float.”
  • an element 700 is held by the element array holder 702.
  • An opening 704 is provided in the element array holder.
  • the opening is sized and shaped to provide a passage through which the proximal end of the element 700 may pass.
  • a top portion 706 is larger than the diameter of the opening 704.
  • the diameter of the opening 704 may be large enough for the element to pass but to limit the degree to which the element may move laterally and angulate.
  • the element “floats” in that it may move upward in direction 708 freely. Gravity pulls the element downward until the top portion 506 rests on the top surface of the element array holder.
  • FIG. 13B The floating is demonstrated in FIG. 13B.
  • the element 700 has been actuated downward to contact the bottom surface 712 of the interior of the well 710. Since, the element 700 floats, the element 700 moves upward in direction 708 as shown so that the top portion of the element 700 no longer is resting of the top surface of the element array holder 702.
  • the floating prevents the element from being damage and also prevents the well 710 from being damaged when the distal end of the element 500 contacts the bottom surface 712 of the well 710.
  • Each element of the element array may be configured to float in this fashion.
  • other mechanisms such as attachment of elements to a spring to allow some movement of the element when contacting a hard surface, while returning it to its original position after removal, may also be used.
  • FIG. 14 depicts an example of a reaction plate 800 that has an array of wells 802 organized in a grid.
  • the reaction plate 800 has an 8 x 12 grid of wells 802, for a total of 96 wells 802. Reaction plates with fewer or additional wells may be used in the exemplary embodiments.
  • Each well 802 may be conical or cylindrical in shape with a circular opening 804. As mentioned above, at least some of the wells 802 may hold liquid reagent volumes, wash solution, deblocking solution, or quenching solution in some exemplary embodiments.
  • FIG. 15 depicts a portion of a patterned surface plate 900 that may be used in exemplary embodiments.
  • the patterned surface plate 900 may, for example, have hydrophilic regions like the circular regions 902 shown in FIG. 15.
  • the patterned surface plate 900 also may include hydrophobic regions 904 that surround the hydrophilic regions 902.
  • a wash solution, a deblocking solution, or a quenching solution also may be applied to such a patterned surface and stay resident on the hydrophilic regions 902.
  • a solution that has an affinity for hydrophobic regions may be dispensed on to the surface of the patterned surface.
  • the pattern on the patterned surface 900 may be reversed so that the regions that are hydrophobic and the regions that are hydrophilic may be reversed (i.e., regions 902 would be hydrophobic and regions 904 would be hydrophilic).
  • FIG. 16 depicts a flowchart 1000 of illustrative steps that may be performed by the synthesis system 100 in exemplary embodiments to synthesize a polymer.
  • one or more surfaces on at least some of the elements in the element array 102 are processed so that the one or more surfaces of the elements become synthesis surfaces that are configured to act as sites where synthesis of portions of the polymer may take place.
  • the polymer is an oligonucleotide
  • portions of the polymer are added iteratively by dipping the elements into different polymer extension solutions comprising a predetermined monomer and a polymerase in a predefined sequence as described below.
  • the synthesis surface may be processed to have the one or more synthesis surfaces functionalized with a surface-bound DNA tag.
  • hybridization is performed so that a starter sequence having a sufficiently complementary sequence is hybridized to a surface bound DNA tag.
  • FIG. 17 depicts operations 1100, 1102, and 1104 that may be performed in some exemplary embodiments as part of the hybridization 1004.
  • the dipping elements array may be washed by a washer.
  • the washed dipping elements may be immersed into a hybridization buffer so that the starter sequence is hybridized to the surface-bound DNA tag.
  • the dipping elements array is washed again by the washer.
  • the hybridization 1004 need not be performed.
  • the depiction of this operation in FIG. 16 is in phantom form.
  • the starter sequence may be directly bound to the surface through a 5' amine functional group. This surface bound starter can be hybridized for quality control purposes or to prevent base-pairing between the starter sequence and the growing nucleic acid oligo on the surface, but hybridization is not necessary for the extension of the tag with a nucleotide since the surface-bound starter has an exposed 3' hydroxyl.
  • FIG. 18 depicts a flowchart of illustrative steps that may be performed in exemplary embodiments in synthesis processing.
  • the synthesis processing is iterative and has numerous cycles.
  • a cycle represents the portion of the processing where a portion of the polymer is attached to the elements of the element array 102.
  • each cycle attaches a blocked nucleotide to the elements.
  • the next cycle is initiated.
  • processing for the cycle is performed as described below.
  • a check is made whether the last cycle has been processed.
  • FIG. 19 depicts a flowchart 1300 of illustrative steps that may be performed in exemplary embodiments during processing for a single cycle.
  • polymer extension solution(s) is/are dispensed into the wells of a plate or locations on a patterned surface by dispenser(s) 126.
  • a single liquid polymer extension solution may be dispensed to all of the wells or locations or different respective polymer extension solutions may be dispensed among corresponding subsets of the wells or locations. For example, as shown in FIG.
  • wells in region 1402 may be filled with a first polymer extension solution comprising a first monomer
  • wells in region 1404 i.e., columns 7-12
  • wells in region 1406 i.e., columns 13-18
  • wells in region 1408 i.e., columns 19-24
  • wells in region 1408 i.e., columns 19-24
  • the wells of the plates or the hydrophilic locations on a patterned surface plate are filled with the desired polymer extension solution depending on the desired sequence of the product on each dipping element.
  • the elements may be dipped into the wells of the next successive region 1402, 1404, 1406 or 1408.
  • the wells or locations on the patterned surface can either be prefilled or filled in-line by a high throughput dispenser (such as the Formulatrix Tempest).
  • a high throughput dispenser such as the Formulatrix Tempest.
  • the synthesis surfaces of the elements are placed in contact with the liquid reagent volumes in the wells or locations.
  • the synthesis surfaces are at least partially immersed in the liquid reagent volumes for a specified period of time.
  • the contact may be achieved by the actuator(s) 106 actuating the actuatable components 109 to move the element array holder 104, the reaction plate or patterned surface 128 or both of those items.
  • the resulting movement caused by the actuation may be in the X, Y and/or Z direction, such as in the configurations 200 and 300 of FIG. 8 or FIG. 9, or may include rotational or angular movement.
  • the synthesis system 100 may contact the elements with the liquid reagent volumes (e.g., polymer extension solution) at the resolved loci for a sufficient time for monomer addition. During this reaction time, all of the elements may be immersed in the liquid reagent volumes simultaneously.
  • the element array 102 can be formatted to match a subset of the wells or hydrophilic locations. For example, a 1536 well reaction plate may be filled with four monomer coupling steps, and used with an array of 384 dipping elements with a 4.5 mm pitch. Similarly, a lower throughput system using 8, 16, 24 or any subset of 1536 dipping elements may be aligned in an array to access wells of a 96, 384, or 1536 reaction well plate or locations on a patterned surface.
  • the plate or patterned surface may be heated by a thermal controller 130.
  • the reaction plates or patterned surfaces 128 also may be chilled by the thermal controller 130 as a means of increasing stability before they are needed for a coupling step.
  • a Peltier may be used to rapidly control plate temperature.
  • cold and warm stations may serve as the thermal controller 130 to change the temperature of the reagents in the reaction plates or patterned surfaces.
  • Preformatted reaction plates or patterned surfaces also may be stored in a cold cabinet for several days at a time.
  • agitation within the coupling well or location can be achieved by plunging the element into the well or location multiple times. Agitation may also be achieved by using a shaker 132, such as an orbital shaker or orbital stage motion, to move the reaction plate or patterned surface with respect to the dipping elements.
  • a shaker 132 such as an orbital shaker or orbital stage motion
  • the elements that were placed in contact with the polymer extension solution(s) may be placed in contact with a quenching solution for quenching the reaction.
  • a quenching solution for quenching the reaction.
  • the same elements may be placed in contact with a deblocking solution, such as proteinase K (ProK).
  • ProK proteinase K
  • These stations can be recirculating, continuously flowing, or static baths.
  • the ProK cleavage step may be heated in order to increase the enzymatic reaction rate.
  • the elements are washed.
  • the washing of the elements can be carried out in dedicated stations of the synthesis system 100, such as described above.
  • the reaction plate or patterned surface may be washed with the plate washer 122 and reloaded with new polymer extension solution in order to save plasticware space and cost. For example, if using an array of 384 elements with a 1536 reaction well plate, the wash cycle may be every four coupling steps.
  • the present disclosure provides a substrate having a functionalized surface.
  • the substrate having a functionalized surface can comprise a solid support having a plurality of resolved loci.
  • the resolved loci are functionalized with a moiety that increases the surface energy of the solid support.
  • the resolved loci are localized on microchannels.
  • surface modifications are employed for the chemical and/or physical alteration of a surface by an additive or subtractive process to change one or more chemical and/or physical properties of a substrate surface or a selected site or region of a substrate surface.
  • surface modification may involve (1) changing the wetting properties of a surface, (2) functionalizing a surface, i.e., providing, modifying or substituting surface functional groups, (3) defunctionalizing a surface, i.e., removing surface functional groups, (4) otherwise altering the chemical composition of a surface, e.g., through etching, (5) increasing or decreasing surface roughness, (6) providing a coating on a surface, e.g., a coating that exhibits wetting properties that are different from the wetting properties of the surface, and/or (7) depositing particulates on a surface.
  • the substrate surface, or the resolved loci, onto which the oligonucleotides or other moieties are deposited may be smooth or substantially planar, or have irregularities, such as depressions or elevations.
  • the surface may be modified with one or more different layers of compounds that serve to modify the properties of the surface in a desirable manner.
  • modification layers of interest include: inorganic and organic layers such as metals, metal oxides, polymers, small organic molecules and the like.
  • Polymeric layers of interest include layers of: peptides, proteins, nucleic acids or mimetics thereof (for example, peptide nucleic acids and the like); polysaccharides, phospholipids, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyetheyleneamines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, and the like, or any other suitable compounds described herein or otherwise known in the art, where the polymers may be hetero- or homopolymeric, and may or may not have separate functional moieties attached thereto (for example, conjugated).
  • Other materials and methods for surface modification of the substrate or coating of the solid support are described in U.S. Pat. No. 6,773,888 and U.S. Pub. No. 2007/0054127, which are herein incorporated by reference in their entirety.
  • the surface of the substrate can also be prepared to have a low surface energy using any method that is known in the art. Lowering the surface energy can facilitate oligonucleotides to attach to the surface.
  • the surface can be functionalized to enable covalent binding of molecular moieties that can lower the surface energy so that wettability can be reduced. In some embodiments, the functionalization of surfaces enables an increase in surface energy and wettability.
  • the polynucleotide synthesis system may be enclosed in chambers with controlled humidity, air content, vapor pressure, and/or pressure forming an assembly with a controlled environment.
  • the humidity of the chambers can be saturated or about 100% to prevent liquid evaporation from the resolved reactors during the reactions.
  • the humidity can be controlled to about, less than about, or more than about 100%, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30% or 25%.
  • the system may comprise a heating component, a cooling component, or a temperature-controlled element (e.g., a thermal cycling device).
  • a thermal cycling device for use with a plurality of resolved reactors may be configured to perform nucleic acid amplification or assembly, such as PCR or PCA or any other suitable nucleic acid reaction described herein or known in the art.
  • the temperature can be controlled such that the temperatures within the reactors can be uniform and heat can be conducted quickly.
  • the systems described herein may have detection components for end-point or real-time detection from the reactors or the individual microstructures within substrates, for example during oligonucleotide synthesis, gene assembly or nucleic acid amplification.
  • Any of the systems described herein may be operably linked to a computer and may be automated through a computer either locally or remotely.
  • Computers and computer systems for the control of the system components described herein are further described elsewhere herein.
  • synthesis of a polynucleotide comprises adding nucleotides stepwise to a synthesis initiator (e.g., an initial oligonucleotide) via the cycled steps of: addition of polymerase-nucleotide conjugate to an oligonucleotide, binding of the nucleotide to the 3' end of the oligonucleotide catalyzed by the polymerase, and cleavage of the polymerase from the added nucleotide. These steps can be repeated until a desired polynucleotide is synthesized.
  • a synthesis initiator e.g., an initial oligonucleotide
  • synthesis of a polynucleotide comprises adding nucleotides stepwise to a starter molecule (e.g., an initial oligonucleotide) via the cycled steps of: addition of polymerase-nucleotide conjugate to an oligonucleotide, binding of the nucleotide to the 3' end of the oligonucleotide catalyzed by the polymerase, and cleavage of the polymerase from the added nucleotide. These steps can be repeated until a desired polynucleotide is synthesized.
  • a starter molecule e.g., an initial oligonucleotide
  • the present disclosure includes a method of synthesizing a polynucleotide comprising contacting a precursor polynucleotide with a nucleotide and a polymerase, wherein said nucleotide comprises comprising a protecting group bound to a base pairing oxygen or nitrogen on the nucleobase.
  • the method of synthesizing a polynucleotide comprises removing a blocking group, such as a conjugated polymerase or a reversible terminator, after addition of a nucleotide to a precursor polynucleotide.
  • the method of synthesizing a polynucleotide comprises repeating contacting, adding, and optionally removing a blocking group described herein one or more times.
  • removal of one or more protecting groups described herein comprises exposing said polynucleotide to a chemical or photolytic condition capable of removing said one or more protecting groups from said protected nucleobases.
  • articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
  • Extension reactions were performed with the following reagents: 250 nM T35 DNA (SEQ ID NO: 48) oligo with 5' fluorescein and purchased from IDT, lx Cutsmart Buffer (New England Biolabs), 0.25 mM CoC12 (New England Biolabs), 1 units/ ⁇ L of terminal transferase (New England Biolabs), 1 mM of "dG” (deoxy guano sine triphosphate) or 1 mM of "06m-dG” (O6-Methyl-deoxy guanosine triphosphate).
  • FIG. 21 shows the results of extension reactions performed for 30 seconds, 1 minute, 2 minutes, 4 minutes, or 8 minutes with dG (left column) or 06m-dG (right column).
  • Starter is unmodified T35 starter oligo (SEQ ID NO: 48).
  • the x-axis is the approximate oligo length in nucleotides and the y-axis is relative fluorescence of fluorescein at 517 nm.
  • Extension reaction with TdT and dGTP produces short products where extension stalls (as G-quadruplexes form).
  • extension with dGTP having an alkylation at the 06-position shows robust and continuous extension without stalling.
  • modified nucleotides such as O6-methyl dGTP, improves polynucleotide synthesis and inhibits secondary structure formation.
  • TdT expression was performed using BL21 (DE3) Gold cells (Agilent) in TB media containing antibiotics for resistance marker of the plasmid.
  • An overnight culture of 50 mL was used to inoculate a 400 mL expression culture with 1/20 vol. Cells were grown at 37° C. and 200 rpm shaking until they reached OD 0.6.
  • IPTG was added to a final concentration of 0.5 mM and the expression was performed for 16-20 h at 16° C.
  • Cells were harvested by centrifugation at 8000 G for 10 min and resuspended in 20 mL buffer A (20 mM Tris-HCl, 0.5 M NaCl, pH 8)+5 mM imidazole.
  • Cell lysis was performed using sonication followed by centrifugation at 30,000 G for 20 min. The supernatant was applied to a gravity column containing 1 mL of Ni-NTA agarose (Qiagen). The column was washed with 20 volumes of buffer A+40 mM imidazole, and bound protein was eluted using 4 mL buffer A+500 mM imidazole.
  • the protein was concentrated to ⁇ 0.15 mL with Vivaspin 20 columns (MWCO 10 kDa, Sartorius) and then dialyzed against 200 mL TdT storage buffer (100 mM NaCl, 200 mM K2HPO4, pH 6.5) overnight using Pur-A-LyzerTM Dialysis Kit Mini 12000 tubes (Sigma).
  • Ni-purified sample was applied to a HiTrap Q HP anion column. Protein was eluted with linear gradient from 100% Q Buffer A (100 mM NaCl, 20 mM K2HPO4, pH 6.5) to 100% Q Buffer B (IM NaCl, 20 mM K2HPO4, pH 6.5). SDS-PAGE analysis was used to identify fractions that contained TdT, these samples were pooled and concentrated.
  • TdT-nucleotide conjugates a cleavable linker-nucleotide with a moiety capable of site specifically conjugating to a cysteine (i.e., maleimide) was first synthesized. Then, equal moles of TdT and linker-nucleotide were incubated overnight at 4°C in 500 mM NaCl, 20 mM K2HPO4, pH 6.5. TdT conjugates were separated from unreacted linker- nucleotide using a S200 size exclusion column (Cytiva) pre-equilibrated in 20 mM Tris Acetate, 50 mM Potassium Acetate; pH 7.9.
  • cysteine i.e., maleimide
  • catalytic divalent metal e.g., cobalt or magnesium at a concentration in the range of 20-10000 pM
  • Both oligos were purchased from ATDBio. Each 6 GC hairpin DNA oligo and TdT- dCTP conjugate were incubated in Tris or HEPES buffer at a pH of 8 and in 50 mM salt (e.g., potassium acetate or NaCl). The reactions were stopped at 3.8 seconds, 6.6 seconds, 11.6 seconds, 19.2 seconds, 29 seconds, 45 seconds, 67 seconds, and 139 seconds by the addition of 40 mM EDTA. Unmodified oligo is labeled as ‘Starter.’ The resulting oligonucleotide reaction products were sized by detecting the fluorescence of the FAM oligo at 517 nm on an ABI 3730x1 DNA Analyzer (FIG. 22).
  • ABI 3730x1 DNA Analyzer FIG. 22
  • the strong hairpin structure of the unmodified 6 GC hairpin (6 base pairing residues of G and C in a row without an alkyl modification) results in limited accessibility of the 3' end of the polynucleotide, thus slowing extension by a polymerase-nucleotide conjugate.
  • a single alkylation disrupts the hairpin structure to increase accessibility of the 3' end of the polynucleotide, leading to substantially increased extension speed. Therefore, the alkylated nucleotide in the hairpin sequence reduces the inhibition of polymerase-nucleotide conjugate addition to a polynucleotide having an adjacent hairpin sequence / secondary structure to its 3' end.
  • the ‘06Bu-G’ sample has all Gs conjugated to TdT via the 06 position. After linker cleavage, hydroxybutyl on the 06 position of G nucleotides are retained, which prevents basepairing.
  • the ‘07Et-G & 06Bu-G’ sample has all 07Et-G, except for two steps of the oligo synthesis, G32 & G44, which contain 06Bu-G bases. These two positions are predicted to have secondary structures within the sequence which can inhibit rapid nucleotide addition by TdT.
  • DNA extension was performed on the starting molecule by cycled addition of nucleotides via TdT-nucleotide conjugates. Each DNA extension cycle to add one nucleotide to the 3' end of the polynucleotide bound to the surface was performed as follows (at a temperature between 24-37°C):
  • TdT-nucleotide conjugate corresponding to either A, T, C, or G
  • divalent metal for instance cobalt, magnesium, for instance at concentrations between 20-10000 pM
  • Tris or HEPES buffer with pH 8 and 50 mM salt for example, potassium acetate or NaCl
  • TdT-nucleotide conjugate After addition of the TdT-nucleotide conjugate to the DNA, the linker connecting the nucleotide and TdT in the conjugate is cleaved using a reagent such as a cleavage enzyme or a reducing agent. 40 mM EDTA is added to terminate the TdT extension reaction.
  • a reagent such as a cleavage enzyme or a reducing agent. 40 mM EDTA is added to terminate the TdT extension reaction.
  • Regeneration of surface A solution of NaOH pH 11 with 0.5 M NaCl was then used to wash away all unbound reaction components from the surface.
  • a reverse primer binding site was added to the 3' end of the synthesized regions of the oligonucleotide synthesis products from Example 4 to allow testing of alkylation of the oligonucleotide via PCR amplification.
  • Addition of a reverse primer binding site, such as addition of a homopolymer tail, is taught, e.g., in Palluk et al. 2018 Nature Biotechnology, and in PCT Publication WO2017/223517, “Nucleic Acid Synthesis and Sequencing Using Tethered Nucleoside Triphosphates,” incorporated by reference in its entirety.
  • hAGT Treatment dealkylation of the synthesized oligonucleotides using human alkylguanine transferase was performed as follows: 200 pM zinc acetate, 20 mM Tris acetate, 50 mM potassium acetate, 400 pM tris(2- carboxyethyl)phosphine, 1 pM human alkylguanine transferase, and 0.1 volumes of adapter ligated synthesis product was added to the synthesized oligonucleotide. Reactions were incubated at 37°C for either 10 minutes, 1 hour, or 4 hours. At each ending timepoint, reactions were inactivated by incubation at 80°C for 10 minutes. Control reactions (“No Treatment”) including the above reagents without human alkylguanine transferase were also performed and terminated at each timepoint.
  • PCR reactions used lx Q5 Master Mix (NEB), 400 nM Forward and Reverse primer, 0.02 volumes of human alkylguanine transferase treated oligonucleotides. Thermocycling of each sample for 40 cycles under standard PCR amplification conditions was performed and the PCR reaction products were run on a 4% Nusieve 3:1 agarose gel (Lonza) at 100 volts for 2 hours and visualized with UV (see FIG. 24).
  • a 50-mer of the same sequence that was synthesized without any alkylations was used as a positive control to demonstrate what size amplification product is expected if the correct PCR amplification occurs.
  • Result As shown in FIG. 24, the synthesis products with the alkylations (e.g., No Treatment) are not amplified, because the polymerase cannot read through the alkyl scars remaining on the polynucleotide. However, treatment of the alkylated 50-mer with AGT enables subsequent PCR amplification, resulting in a PCR product that has the expected size as demonstrated by the comparison with the non-alkylated positive control.
  • Multi-step syntheses were performed to create a 50mer poly-G sequence (SEQ ID NO: 55) expected to form strong G-quadruplex structures (see Example 1)
  • TdT-nucleotide conjugate corresponding to either A, T, C, or G
  • divalent metal for instance cobalt, magnesium, for instance at concentrations between 20-10000 pM
  • Tris or HEPES buffer with pH 8 and 50 mM salt for example, potassium acetate or NaCl
  • TdT-nucleotide conjugate After addition of the TdT-nucleotide conjugate to the DNA, the linker binding the nucleotide and TdT in the conjugate is cleaved using a reagent such as a cleavage enzyme or a reducing agent. 50mM EDTA is added to terminate the TdT extension reaction.
  • a reagent such as a cleavage enzyme or a reducing agent. 50mM EDTA is added to terminate the TdT extension reaction.
  • Regeneration of surface A solution of NaOH pH 11 with 0.5 M NaCl was then used to wash away all unbound reaction components from the surface.
  • Multi-step syntheses were performed to create a defined 40-mer sequence expected to form a 15 bp hairpin
  • TdT-nucleotide conjugate corresponding to either A, T, C, or G
  • divalent metal for instance cobalt, magnesium, for instance at concentrations between 20-10000 pM
  • Tris or HEPES buffer with pH8 and 50mM salt for example, potassium acetate or NaCl
  • TdT-nucleotide conjugate After addition of the TdT-nucleotide conjugate to the DNA, the linker binding the nucleotide and TdT in the conjugate is cleaved using a reagent such as a cleavage enzyme or a reducing agent. 50mM EDTA is added to terminate the TdT extension reaction.
  • a reagent such as a cleavage enzyme or a reducing agent. 50mM EDTA is added to terminate the TdT extension reaction.
  • Regeneration of surface A solution of NaOH pH 11 with 0.5 M NaCl was then used to wash away all unbound reaction components from the surface.
  • An oligonucleotide (5'-FAM-CTGACAGAGATGATGAAGTCACATGAGACATGAACTGAGTC-3') (SEQ ID NO: 36) (was tailed with modified G nucleotides (e.g., O6-methyl G, 06-hydroxypropyl G, 06-hydroxybutyl G, N7-aminomethyl-O6-methyl G, and O6-aminomethylbenzyl G, as shown in FIG. 27).
  • modified G nucleotides e.g., O6-methyl G, 06-hydroxypropyl G, 06-hydroxybutyl G, N7-aminomethyl-O6-methyl G, and O6-aminomethylbenzyl G, as shown in FIG. 27.
  • Tailing was performed as described by NEB (https://www.neb.com/protocols/0001/01/01/a-typical-dna-tailing-reaction).
  • hAGT human AGT
  • TCEP zinc acetate
  • TCEP reducing agent
  • hAGT a dye-labeled, known hAGT substrate, benzyl-guanine- Alexa Fluor 488 (purchased from NEB - product #S9129S)
  • hAGT was separated from free dye by SDS-PAGE and dye-labeled AGT was visualized with UV light. The gel was then stained with Coomassie dye and subsequently imaged as a hAGT loading control.
  • the resulting gel is shown in FIG. 27.
  • the G-methyl hairpin oligo has an alkylation that can react with hAGT, while the G hairpin oligo does not.
  • FIG. 28A and FIG. 28B Screening for an appropriate AGT for dealkylation for an 06-allyl modified G nucleotide was performed for several different species of AGT as shown in FIG. 28A and FIG. 28B.
  • Singly extended oligos were produced by incorporating a polymerase-nucleotide G conjugate onto the 3’ end of an oligonucleotide.
  • Polymerase was separated from the nucleotide by the addition of 0.05 mg/mL thermolabile proteinase K and an incubation at 37°C for 25 minutes. Cleavage of the polymerase left a G nucleotide at the 3’ end comprising an allyl group at the 06 position.
  • De-alkylation was performed using 5 uM of the corresponding AGT species mixed with 20 nM extended oligonucleotide in 400 pM Zn, 1 mM DTT, and IX TP8 buffer.
  • reactions were started by adding the appropriate singly-extended oligo to the corresponding AGT mix and incubating at 37 °C. Reactions were quenched after 1 minute, 6 minutes, 21 minutes, or 60 minutes by the addition of sodium dodecyl sulfate to 0.5%. Oligonucleotides were analyzed for dealkylation of scarred-nucleotide through capillary electrophoresis.
  • FIGs. 28 A and 28B demonstrates the results of the dealkylation reactions.
  • the x-axis represents the approximate oligo length in nucleotides.
  • Scarred singly-extended oligos run to the right of singly-extended oligos with natural nucleotides because of the scars.
  • Allyl-G Oligo in FIG. 28A and 28B shows an untreated oligonucleotide with an O6-allyl modified G nucleotide at the 3’ end (without AGT dealkylation).
  • Extension reactions were performed with the following reagents: a 50 nM polyT DNA oligo sequence containing 35T (SEQ ID NO: 48) or a polyC DNA oligo sequence containing 30C (SEQ ID NO: 50).
  • Each oligo was labeled with a 5’ fluorescein and purchased from IDT, lx TP8 Buffer, metal mix containing 1 mM MgC12 and 0.25 mM CoC12 (New England Biolabs), 1 units/ ⁇ L of terminal transferase (New England Biolabs), 250 pM of "dG” (deoxy guano sine triphosphate), “dA” (deoxyadenosine triphosphate) or modified nucleotide triphosphates.
  • FIGs. 29 A and 29B show extension reactions performed for 30 seconds, 1 minute, 4 minutes, or 8 minutes.
  • the x-axis is the approximate oligo length in nucleotides and the y-axis is relative fluorescence of fluorescein at 517 nm.
  • the red-dashed line in each plot represents the position of the starter.
  • FIG. 29A shows data using the polyT starter with dG (left column), 06Me dG, O6Allyl dG, and O6Bu dG.
  • Extension reaction with TdT and dGTP produces short products where extension stalls (as G-quadruplexes form). In contrast, extension with dGTP containing modified nucleotides shows robust and continuous extension without stalling.
  • FIG. 29B shows an experiment using the oligonucleotide starter containing a sequence of all C’s, TdT and various nucleotides, dG (left column), 06Me dG, O6Allyl dG, and O6Bu dG.
  • the extension reaction with dGTP is quite slow and base-pairing with the C template prevents extension of the starter.
  • nucleotide extension containing 06- position modifications has continuous nucleotide turnover.
  • the use of nucleotides containing modifications at the 06 position in dG improves polynucleotide synthesis and inhibits secondary structure formation.
  • Terminal, singly-extended oligos were produced by using the following reagents: 300 nM starter oligo (5'-6-FAM- CTGACAGAGATGATGAAGTCACATGAGACATGAACTGAGTCTTTT-3' (SEQ ID NO: 34)), 70 mM tris potassium buffer pH 8, 100 pM cobalt-acetate, 6 pM bacterial phosphatase, and 1.5 pM of corresponding conjugate (polymerase-nucleotide conjugate cleavable by proK - after proK cleavage leaves ‘scarred nucleotide’ of allyl-O6 dGTP, butyl(Bu)-O6-dGTP, allyl-O4 dUTP, or propargyl(Prg)-O4 dUTP).
  • reactions were started by adding the appropriate conjugate to the oligo mix and incubating at room temperature for 5 minutes. Reactions were quenched by the addition of 0.5 mM DM-nitrophen. Polymerase was separated from the nucleotide by the addition of 0.05 mg/mL thermolabile proteinase K and an incubation at 37°C for 25 minutes.
  • Thermolabile proteinase K was heat-denatured with a 10-minute incubation at 65 °C. DM- nitrophen was broken down with a 3-minute UV light treatment. Oligonucleotides were analyzed for +1 addition of nucleotide through capillary electrophoresis.
  • FIG. 30 demonstrates the results of the extension reactions as measured by capillary electrophoresis.
  • the x-axis represents the approximate oligo length in nucleotides and the y-axis represents the fluorescence of fluorescein at 517 nm.
  • Starter is unmodified starter oligo as described above.
  • Extension reaction with each of the conjugates leads to a shift to the right compared to the starter oligo, indicating the addition of a scarred nucleotide.
  • TdT conjugated to the nucleotides successfully was added to the starter oligonucleotide at the 3’ end, generating singly-extended substrates. These were then used for the following dealkylation experiments.
  • FIGs. 31A - 31D demonstrate the results of the 60 minute dealkyation reactions.
  • the x-axis represents the approximate oligo length in nucleotides and the y-axis represents the fluorescence of fluorescein at 517 nm. Scarred singly-extended oligos run to the right of singly-extended oligos with natural nucleotides because of the scars.
  • modified nucleotides comprising N-linked protecting groups or scars (as a portion of a linker in a conjugate) used in the examples (and referred to throughout the specification) are provided below in Table 4.
  • the structures shown in Table 4 show the nucleobase-Z-Ll-L2-Rl structure, which represents a protected nucleobase or a scarred nucleobase cleaved from a linker.
  • These structures also describe the Z-L1-L2 portion of the linker in a conjugate, such that the linker comprises a cleavable element that results in the scarred nucleotide shown.
  • the structures in the table are the reactants for the described deprotection / scar removal examples and reaction conditions.
  • Table 4 Exemplary nucleotides with protecting groups / scars linked to O or N base pairing atoms
  • FIG. 32A is an HPLC chromatogram showing the trace of the photocleavable dGTP nucleotide, the starting material for the photocleavage experiment.
  • the X-axis is represented in minutes.
  • the peak eluting at 5.693 minutes is representative of the intact photocleavable dATP nucleotide.
  • FIG. 32B is an HPEC chromatogram showing the trace of native dGTP nucleotide, the expected product of the photocleavage reaction.
  • the X-axis is represented in minutes.
  • the peak eluting at 2.001 minutes is representative of the native dATP nucleotide.
  • FIG. 32C is an HPLC chromatogram showing the trace of the photocleavage reaction product after 120 minutes exposure to a 365 nm wavelength lamp. The X-axis is represented in minutes.
  • FIG. 33A is an HPLC chromatogram showing the trace of the photocleavable dATP nucleotide, the starting material for the photocleavage experiment.
  • the X-axis is represented in minutes.
  • the peak eluting at 5.693 minutes is representative of the intact photocleavable dATP nucleotide.
  • FIG. 33B is an HPLC chromatogram showing the trace of the reaction product of photocleavable dATP after exposure to a 365 nm wavelength lamp for 120 minutes.
  • the X- axis is represented in minutes.
  • the peak eluting at 3.426 minutes is representative of the native dATP nucleotide.
  • TdT can incorporate a nucleotide analog with a photocleavable scar into DNA
  • a photocleavable nitrobenzyl dGTP (06 Nitrobenzyl G - See Table 4) was synthesized and conjugated to TdT to form a nucleotide-polymerase conjugate. An extension reaction was performed and incorporation of the dGTP analog into the extended polynucleotide was analyzed.
  • fluorescently labeled DNA was hybridized to a surface immobilized complementary strand.
  • the hybridized DNA was extended 1 nucleotide as described above and then either removed from its complementary strand with highly deionized formamide or extended again for a total of 2 and then 3 additions.
  • Each extension cycle was initiated with the addition of 0.1 mg/mL of TdT conjugate and allowed to proceed for 6 min in a buffer containing 20 mM Tris Acetate, 50mM Potassium Acetate and 50 uM Cobalt Acetate at pH 7.9.
  • the blocking TdT protein was cleaved from the nucleotide analog, leaving a nitrobenzyl scar on the DNA molecule.
  • a portion of the synthesized oligonucleotides were irradiated with 365 nm light to remove the photocleavable scar. The resulting oligonucleotides were then analyzed using capillary electrophoresis.
  • FIG. 34A Processed capillary electrophoresis pherograms show that the polynucleotide can be extended by 1, 2 and 3 subsequent additions of nitrobenzyl dGTP by the TdT conjugate (FIG. 34A).
  • FIG. 34B shows products from extension reactions containing polynucleotide products irradiated with UV light (365 nm) for 20 min. Comparison of FIG. 34A and FIG.
  • a 45 mer starter DNA sequence was used as the initial polynucleotide upon which the extension reaction was performed.
  • TdT - nucleotide conjugate was prepared for each linker-nucleotide as described herein.
  • the linker was attached to the N4 atom of the cytosine nucleobase.
  • the following protecting groups in the linker were tested (see Table 4 for corresponding linker-nucleotide structures):
  • each of the cytosine conjugates has a peak that is shifted to the right as compared to the starter DNA oligo sample. Therefore, each of the cytosine conjugates successfully extended the starter DNA oligo by incorporating cytosine into the 3' end of the starter oligonucleotide.
  • the presence of two peaks indicates that the linker scar may have already separated from the C nucleotide in a population of the extended oligonucleotides.
  • Example 14 Polynucleotide extension using N-linked A conjugates with chemically removable protecting groups
  • a 45 mer starter DNA sequence was used as the initial polynucleotide upon which the extension reaction was performed.
  • TdT-conjugate was prepared for each linker- nucleotide as described herein. The linker was attached to the N6 atom of the adenine nucleobase. The following conjugates were tested (see Table 4 for corresponding linker- nucleotide structures):
  • reaction products were incubated at ambient temperature for 3 minutes and quenched with the addition of 50 mM Ethylenediaminetetraacetic acid (EDTA) and 0.5 pM ProK (New England Biolabs), which cleaves the polymerase from the nucleotide, leaving the linker scar (i.e., protecting group) on adenine.
  • Reaction products and a control starter DNA oligo sample were then measured using capillary electrophoresis.
  • the reaction product of each of the different adenine conjugates has a peak that is shifted to the right as compared to the starter DNA oligo sample.
  • each of the cytosine conjugates successfully extended the starter DNA oligo by incorporating cytosine into the 3' end of the starter oligonucleotide.
  • the presence of two peaks indicates that the linker scar may have already separated from the A nucleotide in a population of the extended oligonucleotides.
  • Example 15 Polynucleotide extension using O-linked T and U conjugates with chemically removable protecting groups
  • a 45 mer starter DNA sequence was used as the initial polynucleotide upon which the extension reaction was performed.
  • TdT-conjugate was prepared for each linker- nucleotide as described herein. The linker was attached to the 04 atom of the uracil or thymine nucleobase. The following conjugates were tested (see Table 4 for corresponding linker- nucleotide structures):
  • Example 16 Polynucleotide extension using O-linked G conjugates with chemically removable protecting groups
  • a 45 mer starter DNA sequence was used as the initial polynucleotide upon which the extension reaction was performed.
  • TdT-conjugate was prepared for each linker- nucleotide as described herein. The linker was attached to the 06 atom of the guanine nucleobase. The following conjugates were tested (see Table 4 for corresponding linker- nucleotide structures):
  • each of the guanine conjugates has a peak that is shifted to the right as compared to the starter DNA oligo sample. Therefore, each of the guanine conjugates successfully extended the starter DNA oligo by incorporating guanine into the 3' end of the starter oligonucleotide.
  • a dGTP bound to the 06 of guanine (06 sulfone G) was synthesized and conjugated to a TdT variant. Subsequent additions of the same photocleavable sulfone dGTP were performed using the conjugate on the extended DNA to demonstrate that TdT can add additional 06 sulfone G nucleotide analogs to the polynucleotide to form a homopolynucleotide containing the sulfone containing dGTP analog. Finally, the polynucleotide containing the sulfone scar was removed by treatment with an appropriate base (e.g., NH40H) to demonstrate the synthesis of scarless DNA.
  • an appropriate base e.g., NH40H
  • the pH was set to pH 7 or pH 8 at 35°C, and the polynucleotide was incubated for 10 minutes, 45 minutes, or 16 hours to monitor elimination of the sulfone to generate a natural guanine base.
  • the products were analyzed via capillary electrophoresis as shown in FIG. 39.
  • the first row contains a control ssDNA that serves as a marker for where a single natural guanine on the 3' end of the starter oligo migrates. Hashed vertical markers showing the migration of natural guanine and +1 sulfone-guanine are indicated for clarity.
  • Results of the analysis can show that the sulfone dGTP can be incorporated into a polynucleotide in an extension reaction by a TdT conjugate, and that treatment with an appropriate base (i.e., alkaline conditions) removes the sulfone scar, resulting in a native nucleobase.
  • an appropriate base i.e., alkaline conditions
  • the single extension product Starter Oligo + 06 Sulfide G was diluted to 50 nM. This species was then diluted 1:9 in a 10 mM potassium acetate buffer, pH 5.2 which contained 5 mM Oxone, in order to oxidize the sulfide scar to a sulfone scar. The reaction was then incubated at ambient temperature for 10 min after which an aliquot was taken and saved for future analysis. Sodium hydroxide was then added to the reaction to a final concentration of 50 mM in order to fully deprotect the sulfone scar. The reaction was then incubated at 37 degrees Celsius for 1 hour. The fully deprotected product was then compared to a Starter Oligo + dG control, the Starter Oligo + 06 Sulfide G, and the oxidation only control by capillary electrophoresis.
  • the single extension product starter DNA oligo + N6 Carbamate Sulfide A was then exposed to 50mM NaOH at 60°C for 30 minutes or 16 hours.
  • a sample comprising the single extension product, a sample comprising the extension product after 30 minutes exposure to 50mM NaOH, and a sample comprising the extension product after 16 hours exposure to 50mM NaOH was analyzed by capillary electrophoresis. As shown in FIG. 41A the scar on the A nucleotide of the extension product was partially removed after 30 minutes, and completely removed after 16 hours of exposure to 50mM NaOH.
  • the single extension product starter DNA oligo + N4 Carbamate Sulfide C was then exposed to 50mM NaOH at 60°C for 30 minutes or 16 hours.
  • a sample comprising the single extension product, a sample comprising the extension product after 30 minutes exposure to 50mM NaOH, and a sample comprising the extension product after 16 hours exposure to 50mM NaOH was analyzed by capillary electrophoresis. As shown in FIG. 41B the scar on the C nucleotide of the extension product was partially removed after 30 minutes, and completely removed after 16 hours of exposure to 50mM NaOH.
  • the single extension product starter DNA oligo + N6 Carbamate Ethyl A was then exposed to 50mM NaOH at 37°C for 30 minutes.
  • a sample comprising the single extension product and a sample comprising the extension product after 30 minutes exposure to 50mM NaOH was analyzed by capillary electrophoresis. As shown in FIG. 42 the scar on the A nucleotide of the extension product was completely removed after 30 minutes exposure to 50mM NaOH.
  • the single extension products were diluted to 50 nM. These species were then diluted 1:9 in water or 10% triethylamine (aq.) and incubated at 37 degrees Celsius for 30 min. After treatment, all samples were evaporated to dryness and redissolved in water. The treated samples were then compared to the control samples for each scar by capillary electrophoresis.
  • FIG. 43 shows capillary electrophoresis results for i) an oligonucleotide comprising a N6-linked scarred adenine, and ii) the same oligonucleotide after 30 minute treatment with triethylamine (TEA), where the N6-linked scarred adenine are: N6 Carbamate Propyl A, N6 Carbamate Ethyl A, N6 Amide Propyl A, and N6 Amide Ethyl A (see Table 4).
  • TAA triethylamine
  • the results indicate that exposure of a scarred or protected nucleobase comprising a carbamate or amide group linked to an alkyl group of a preferred length to a base results in the cyclization and subsequent removal of the scar, which is more preferred than the nucleophilic or base removal mechanism described above.
  • TEA can be used to remove the scars from each of the scarred nucleotides. Specifically, intramolecular cyclization that forms a 5-membered ring leaving group is favored (faster) for scar deprotection over a 4 or 6-membered ring. This is represented in FIG. 44, showing a reaction mechanism for cleavage of the N-linked carbamate scar preferring a 5-membered ring structure leaving group. A similar reaction mechanism and preference is observed for the N-linked amide scar. Therefore, the structures can be chosen accordingly to select a desired scar / protecting group that has an optimized balance of stability and removal efficiency for a desired application.
  • the single extension products were diluted to 50 nM. These species were then diluted 1:9 in lx TPB buffer (20 mM Tris Acetate, 50 mM Potassium Acetate, pH 8) and incubated at 98 degrees Celsius for 8 min. Aliquots were taken out at each 2 min interval and compared to the starting material by capillary electrophoresis. Percent deprotection was determined by integrating the peaks for the protected starting material and deprotected product. An exponential curve was then fit to the timepoints for each scarred linker nucleotide and used to determine the associated scar deprotection half-life under these conditions.
  • FIG. 45 shows percent of deprotected oligonucleotides at 2, 4, 6, 8 and 10 minutes for N6 Carbamate Ethyl A (large circles; Et-CO2-A) and N6 Amide Propyl A (squares, Pr- CO-A).
  • N6 Carbamate Ethyl A had a deprotection half-life of 30.8 minutes, while N6 Amide Propyl A had a deprotection half-life of 9.2 minutes.
  • N6 Carbamate Ethyl A was 99.99% deprotected after 409 minutes, while N6 Amide Propyl A was 99.99% deprotected after 123 minutes. Therefore, the amide scar was removed faster than the corresponding carbamate scar having the same 5-membered ring leaving group.
  • N4 Carbamate Ethyl C contains a methyl-substituted hydrogen on the 5-membered ring leaving group (see structure in Table 4).
  • the single extension products were diluted to 50 nM. These species were then diluted 1:9 in lx TPB buffer (20 mM Tris Acetate, 50 mM Potassium Acetate, pH 8) and incubated at 98 degrees Celsius for 8 min. Aliquots were taken out at each 2 min interval and compared to the starting material by capillary electrophoresis. Percent deprotection was determined by integrating the peaks for the protected starting material and deprotected product. An exponential curve was then fit to the timepoints for each scarred linker nucleotide and used to determine the associated scar deprotection half-life under these conditions.
  • FIG. 45 shows percent of deprotected oligonucleotides at 2, 4, 6, 8 and 10 minutes for N4 Carbamate Ethyl C (triangles; Et-CO2-C) and N4 Carbamate (Methyl) Ethyl C (small circles, 2MeEt-CO2-C).
  • N4 Carbamate Ethyl C had a deprotection half-life of 13.5 minutes, while N4 Carbamate (Methyl) Ethyl C had a deprotection half-life of 4.0 minutes.
  • N4 Carbamate Ethyl C was 99.99% deprotected after 179 minutes, while N4 Carbamate (Methyl) Ethyl C was 99.99% deprotected after 53 minutes.
  • Extension reactions were performed with the following reagents: 50 nM starter oligonucleotide, lx TP8 Buffer, metal mix containing 1 mM MgC12 and 0.25 mM
  • FIG. 46A, FIG. 46B, and FIG. 47 show extension reactions performed for 30 seconds, 1 minute, 4 minutes, or 8 minutes and analyzed by capillary electrophoresis.
  • the x- axis is the approximate oligo length in nucleotides and the y-axis is relative fluorescence of fluorescein at 517 nm.
  • the red-dashed line in each plot represents the position of the starter.
  • FIG. 46A shows data using the 35T homopolymer starter oligo (SEQ ID NO: 48) with dG (left column), 06 Sulfone G, and 06 Sulfide G.
  • Extension reaction with TdT and dGTP produces short products where extension stalls (as G-quadruplexes form).
  • extension with dGTP containing modified nucleotides that block 06 hydrogen bonding shows robust and continuous extension without stalling.
  • FIG. 46B shows an experiment the 30C homopolymer starter oligo (SEQ ID NO: 50), TdT and various nucleotides, dG (left column), 06 Sulfone G, or 06 Sulfide G.
  • the extension reaction with dGTP is quite slow and base-pairing with the C template prevents extension of the starter.
  • nucleotide extension containing 06-position modifications has continuous nucleotide turnover.
  • Nucleoside analogue (9/11/12/13, 1 eq) was placed into a 10 mL round bottom flask equipped with a stir bar and tetrabutylammonium pyrophosphate was placed in a separate 5 mL conical tube.
  • the two flasks were placed in a vacuum desiccator with P2O5 and allowed to dry under vacuum for at least 16 hr.
  • molecular sieves and three small round bottom flasks were placed in a drying oven for at least 16 hr. Two small flasks from the oven were charged with molecular sieves and flame-activated under vacuum. While these were cooling, the other small flask was attached to a Hickman distillation apparatus and flame dried.
  • Trimethyl phosphate and tributyl amine were then placed over the molecular sieves in the initial two flasks for drying.
  • the Hickman distillation apparatus was then used to freshly distill POCh.
  • the vacuum desiccator was purged with N2 gas, and the flasks inside were then transferred to nitrogen balloons or the Schlenk line.
  • Trimethyl phosphate (40 eq) was added to the nucleoside and the mixture was cooled at -5 °C.
  • To this nucleoside mixture was added dry tributyl amine (3 eq) followed by POCh (2.1, 1.3, 1.5, 1.8 eq respectively) slowly via micro syringe.
  • the aqueous layer was then purified by reverse phase HPLC (0.1 M triethylammonium acetate buffer/ Acetonitrile, 4- 47%, 0-15 min, flow 5 ml min' 1 ). Product containing fractions were pooled and lyophilized to provide desired product as a triethylammonium salt. The resulting solid was reconstituted in RNase Free DI water for further experiments.
  • TdT expression was performed using BL21 (DE3) Gold cells (Agilent) in TB media containing antibiotics for resistance marker of the plasmid.
  • An overnight culture of 50 mL was used to inoculate a 400 mL expression culture with 1/20 vol. Cells were grown at 37° C. and 200 rpm shaking until they reached OD 0.6.
  • IPTG was added to a final concentration of 0.5 mM and the expression was performed for 16-20 h at 16° C.
  • Cells were harvested by centrifugation at 8000 G for 10 min and resuspended in 20 mL buffer A (20 mM Tris-HCl, 0.5 M NaCl, pH 8)+5 mM imidazole.
  • Cell lysis was performed using sonication followed by centrifugation at 30,000 G for 20 min. The supernatant was applied to a gravity column containing 1 mL of Ni-NTA agarose (Qiagen). The column was washed with 20 volumes of buffer A+40 mM imidazole, and bound protein was eluted using 4 mL buffer A+500 mM imidazole.
  • the protein was concentrated to ⁇ 0.15 mL with Vivaspin 20 columns (MWCO 10 kDa, Sartorius) and then dialyzed against 200 mL TdT storage buffer (100 mM NaCl, 200 mM K2HPO4, pH 6.5) overnight using Pur-A-LyzerTM Dialysis Kit Mini 12000 tubes (Sigma).
  • Ni-purified sample was applied to a HiTrap Q HP anion column. Protein was eluted with linear gradient from 100% Q Buffer A (100 mM NaCl, 20 mM K2HPO4, pH 6.5 ) to 100% Q Buffer B (IM NaCl, 20 mM K2HPO4, pH 6.5). SDS-PAGE analysis was used to identify fractions that contained TdT, these samples were pooled and concentrated.
  • TdT-nucleotide conjugates a cleavable linker-nucleotide with a moiety capable of site specifically conjugating to a cysteine (i.e., maleimide) was first synthesized. Then, equal moles of TdT and linker-nucleotide were incubated overnight at 4°C in 500 mM NaCl, 20 mM K2HPO4, pH 6.5. TdT conjugates were separated from unreacted linker- nucleotide using a S200 size exclusion column (Cytiva) pre-equilibrated in 20 mM Tris Acetate, 50 mM Potassium Acetate; pH 7.9.
  • cysteine i.e., maleimide
  • the ester-containing linker leaving an alcohol scar on the nucleotide is acceptable for DNA synthesis.
  • the alcohol generated by the ester cleavage is a charge-neutral cleavage product, allows for unperturbed nucleotide addition, and is an improvement over protease cleavage which leaves charged nucleotide scars that negatively impact oligo synthesis.
  • An ssDNA primer was extended for 60 seconds with 1) a Linker 1 conjugate, 2) a Linker 2 conjugate, 3) a Linker 2 conjugate (replicate), and 4) no conjugate.
  • TdT-dTTP conjugates containing either linker formed a covalent primer-extension complex with >95% yield in under a minute, as measured by a gel shift assay on SDS-PAGE (EIG. 50).
  • T/P TdT/DNA complex.
  • P ssDNA primer (unbound). Bands above the complex are products with more than 1 base added to the primer.
  • the conjugates comprising amino acid ester linkers 1 and 2 are successfully added to the 3' end of the oligonucleotide and are suitable for oligonucleotide synthesis. Esterase Screening and Enzymatic Cleavage
  • amino acid ester-containing linkers in the polymerase-nucleotide conjugates which have already shown to have good oligonucleotide incorporation kinetics, can also be successfully cleaved using a protease comprising an esterase activity.
  • dTTP conjugates comprising linker 2 were stored overnight at pH 6.5, pH 7.0, pH 7.5, pH 8.0, pH 8.5, or in buffer only as a control.
  • a primer extension was performed incorporating the incubated dTTP conjugates into a primer.
  • the resulting extension product was then run on a capillary electropherogram. The results are shown in FIG. 51, with insertions indicated by the second peak at ⁇ 59. Insertions indicate the presence of free dNTPs in the conjugate solution.
  • Linker 2 conjugates release dNTPs when stored at pH > 6.5. However, at pH 6.5, we found a significantly reduced release of nucleotides by the conjugate.
  • dT(10) SEQ ID NO: 54
  • dT(100) SEQ ID NO: 51
  • dT(200) SEQ ID NO: 52
  • Example 26 A, C, T, and G conjugates with protease ester cleavable linker

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente divulgation concerne, entre autres, des procédés de synthèse de longs polynucléotides, comprenant des procédés améliorés de synthèse de polynucléotides qui peuvent atteindre un rendement par étape élevé et/ou de faibles taux d'erreur, y compris pour de longs polynucléotides, à l'aide de techniques de synthèse de novo indépendante d'un modèle par étapes.
PCT/US2024/035137 2023-06-21 2024-06-21 Synthèse de novo indépendante d'un modèle par étapes de polynucléotides longs Pending WO2024264000A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363509522P 2023-06-21 2023-06-21
US63/509,522 2023-06-21

Publications (2)

Publication Number Publication Date
WO2024264000A2 true WO2024264000A2 (fr) 2024-12-26
WO2024264000A3 WO2024264000A3 (fr) 2025-05-15

Family

ID=93936334

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/035137 Pending WO2024264000A2 (fr) 2023-06-21 2024-06-21 Synthèse de novo indépendante d'un modèle par étapes de polynucléotides longs

Country Status (1)

Country Link
WO (1) WO2024264000A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025155732A1 (fr) * 2024-01-16 2025-07-24 Ansa Biotechnologies, Inc. Procédés et compositions améliorés pour clonage des polynucléotides synthétisés de novo

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7897737B2 (en) * 2006-12-05 2011-03-01 Lasergen, Inc. 3′-OH unblocked, nucleotides and nucleosides, base modified with photocleavable, terminating groups and methods for their use in DNA sequencing
EP4276198B1 (fr) * 2016-06-24 2025-11-26 The Regents of the University of California Synthèse et séquençage d'acide nucléique en utilisant des nucléosides triphosphates fixés
US20190078126A1 (en) * 2017-09-08 2019-03-14 Sigma-Aldrich Co. Llc Polymerase-mediated, template-independent polynucleotide synthesis
US20220145295A1 (en) * 2018-10-12 2022-05-12 President And Fellows Of Harvard College Enzymatic rna synthesis
US11702683B2 (en) * 2020-05-28 2023-07-18 Microsoft Technology Licensing, Llc De novo polynucleotide synthesis with substrate-bound polymerase

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025155732A1 (fr) * 2024-01-16 2025-07-24 Ansa Biotechnologies, Inc. Procédés et compositions améliorés pour clonage des polynucléotides synthétisés de novo

Also Published As

Publication number Publication date
WO2024264000A3 (fr) 2025-05-15

Similar Documents

Publication Publication Date Title
US11624079B2 (en) Polymerase-tethered nucleotides for nucleic acid synthesis
JP7119041B2 (ja) Ｄｎａ配列において標識されたヌクレオチドを検出する方法
US7544794B1 (en) Method for sequencing DNA and RNA by synthesis
JP2022521094A (ja) 共転写キャッピング用ｒｎａポリメラーゼバリアント
CN111051512A (zh) 非天然核苷酸的掺入及其方法
KR20240055718A (ko) 조작된 폴리머라제
US8153779B2 (en) Nucleotide with an alpha-phosphate mimetic
WO2024264000A2 (fr) Synthèse de novo indépendante d'un modèle par étapes de polynucléotides longs
CN120500533A (zh) 用于将聚合酶栓系至核苷酸的可裂解接头
US20250197820A1 (en) Nucleic acid polymerase and its use in producing non-dna nucleotide polymers
HK40078325A (en) Nucleic acid synthesis and sequencing using tethered nucleoside triphosphates
CA3240455A1 (fr) Compositions pour synthese de polynucleotides enzymatiques et procedes d'utilisation
Fraley The synthesis and employment of nucleoside triphosphate analogues as probes for DNA polymerase interactions

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24826787

Country of ref document: EP

Kind code of ref document: A2