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WO2024256604A1 - Addition de sels pendant la synthèse enzymatique de polynucléotides - Google Patents

Addition de sels pendant la synthèse enzymatique de polynucléotides Download PDF

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Publication number
WO2024256604A1
WO2024256604A1 PCT/EP2024/066486 EP2024066486W WO2024256604A1 WO 2024256604 A1 WO2024256604 A1 WO 2024256604A1 EP 2024066486 W EP2024066486 W EP 2024066486W WO 2024256604 A1 WO2024256604 A1 WO 2024256604A1
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reaction site
polynucleotide
salt
extension
initiator
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Henri Lachaize
Ludivine PROFIT
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DNA Script SAS
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DNA Script SAS
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1264—DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal nucleotidyl transferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07031—DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal deoxynucleotidyl transferase

Definitions

  • Nucleic acids are highly charged polyanionic molecules.
  • the metal ions in the solution such as Na + and Mg 2+ ions, play an essential role in stabilizing the folded structure through electrostatic screening.
  • Single stranded junctions and loops between helices such as hairpin structures are important structural and functional components of nucleic acids.
  • methods of the invention include methods of enzymatically synthesizing at least one polynucleotide, the method comprising: (a) providing at least one reaction site; (b) providing at least one initiator at the reaction site, wherein each initiator comprises a free 3'- hydroxyl group; (c) contacting the initiator with a 3’-O-protected nucleoside triphosphate and a template-free polymerase under suitable conditions for enzymatic extension of the initiator, wherein incorporation of the 3’-O-protected nucleoside triphosphate results in production of a 3’- O-protected extension product at each of the reaction sites; (d) performing one or more cycles of i) deprotecting the 3 ’-0 -protected extension products at the reaction site, wherein extension products having free 3 ’-hydroxyl groups are formed at the reaction site, and ii) contacting the reaction site with a 3’-O-protected nucleoside triphosphate
  • kits for enzymatically synthesizing a polynucleotide comprising at least one 3’-O-protected nucleoside triphosphate, a template-free polymerase and at least one salt compound.
  • Fig. 1 illustrates the basic steps of template-free enzymatic synthesis of polynucleotides in this invention.
  • Fig. 2A and Fig. 2B illustrate the comparison of purity of sequences Nl, N2, N4, N6 with different NaCl concentration in mM and different addition time in sec.
  • the practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, molecular biology (including recombinant techniques), cell biology, and biochemistry, which are within the skill of the art.
  • conventional techniques may include, but are not limited to, preparation and use of synthetic peptides, synthetic polynucleotides, monoclonal antibodies, nucleic acid cloning, amplification, sequencing and analysis, and related techniques. Protocols for such conventional techniques can be found in product literature from manufacturers and in standard laboratory manuals, such as Genome Analysis: A Laboratory Manual Series (Vols.
  • Methods of the invention include methods of enzymatically synthesizing at least one polynucleotide, the method comprising: (a) providing at least one reaction site; (b) providing at least one initiator at the reaction site, wherein each initiator comprises a free 3'-hydroxyl group; (c) contacting the initiator with a 3 ’ -O-protected nucleoside triphosphate and a template-free polymerase under suitable conditions for enzymatic extension of the initiator, wherein incorporation of the 3 ’-O-protected nucleoside triphosphate results in production of a 3’-O- protected extension product at each of the reaction site; (d) performing one or more cycles of i) deprotecting the 3’-O-protected extension products at the reaction site, wherein extension products having free 3 ’-hydroxyl groups are formed at the reaction site, and ii) contacting the reaction site with a 3’-O-protected nucleoside triphosphate and a template-
  • the at least one salt compound is added at the reaction site during the extension step of step (c). In some embodiments, the at least one salt compound is added at the reaction site during the extension step (ii) of step (d), in particular during one or more or all of the extension steps (ii) of step (d). In some embodiments, the at least one salt compound is added at the reaction site during the extension step of step (c) and during the extension step of step (d).
  • the at least one polynucleotide, the at least one reaction site and the least one initiator are a plurality of polynucleotides, a plurality of reaction sites and a plurality of initiators.
  • at least one of the plurality of polynucleotides comprises a secondary structure, such as a hairpin structure and/or one or more hard codons.
  • the present invention encompasses the use of various salt compounds.
  • template-free polymerases such as TdT
  • TdT template-free polymerases
  • various salts which can be used in the present invention, e.g. magnesium salts, sodium salts or potassium salts, such as magnesium acetate, magnesium chloride, potassium acetate or potassium chloride.
  • the at least one salt compound comprises an alkali metal or alkaline earth metals salt, preferably an alkali metals salt, more preferably an alkali metals salt chosen among the group of lithium and sodium.
  • the salt comprises sodium, such as sodium chloride salt or lithium chloride salt.
  • the at least one salt compound is one salt compound.
  • the template-independent DNA polymerase is a terminal deoxynucleotidyl transferase (TdT).
  • the pH during the extension step ii) of (d) is comprised between 5- 10, preferably between 6 and 9, more preferably between 6,5 and 8.
  • the at least one salt compound concentration after addition at the reaction site is comprised between 20 millimolar (mM) and 1 M, preferably between 20 millimolar and 500 mM, still preferably between 30 mM and 400 mM, more preferably between 40 and 300 mM, even more preferably between 50 and 200 mM
  • the at least one salt compound concentration after addition at the reaction site is comprised between 20 millimolar (mM) and 500 mM, preferably between 30 mM and 500 mM, more preferably between 50 mM and 500 mM, even more preferably between 100 mM and 500 mM.
  • the at least one salt compound concentration after addition at the reaction site is of at least 20 mM, 50 mM, 100 mM, 150 mM, 200 mM, or 300 mM.
  • one or more salts are present at the reaction site before addition of the salt compound, e.g. in the elongation buffer, and the addition of the at least one salt compound increases the total salt concentration at the reaction site by at least 20%, preferably at least 30%, still preferably at least 40%, even more preferably at least 50%, most preferably at least 100%.
  • said salts present at the reaction site before addition of the salt compound may comprise cacodylic acid salt.
  • the total salt concentration at the reaction site during the extension step and before addition of the at least one salt compound does not exceed 500 mM, preferably 300 mM, more preferably 200 mM, still more preferably 100 mM.
  • said salts present at the reaction site before addition of the salt compound may comprise cacodylic acid salt.
  • the buffer concentration e.g. pH buffer concentration
  • the buffer present at the reaction site may comprise cacodylic acid salt.
  • the addition of the salt compound is realized after at least 20% of the elongation time, preferably after at least 30%, more preferably after at least 50%, even more preferably after at least 70% of the elongation time.
  • the addition of the salt compound is realized at least 10 seconds after the beginning of elongation, preferably at least 20 seconds, more preferably at least 50 seconds, still more preferably after at least 80 seconds, even more preferably after at least 120 seconds, most preferably after at least 190 seconds after the beginning of elongation.
  • the beginning of elongation corresponds to the time at which the reaction site is contacted with the 3’- O-protected nucleoside triphosphate and the template-free polymerase, in particular through the addition of the 3 ’-O-protected nucleoside triphosphate and the template-free polymerase at the reaction site.
  • the addition is done only if the polynucleotide comprises no hard codon.
  • the addition is done only if the polynucleotide comprises at least one secondary structure, preferably a hairpin structure.
  • the polynucleotide is capable to form at least one secondary structure, preferably a hairpin structure.
  • the polynucleotide comprises at least one hard codon.
  • the polynucleotide comprises at least one secondary structure, preferably a hairpin structure and at least one hard codon.
  • the at least one reaction site is on a solid support and during the step (b), at least one initiator is immobilized on the surface of the solid support.
  • the method comprises a final step of deprotection of the 3’-0 protected terminal nucleotide.
  • the synthesized polynucleotide comprise a 3’-0 deprotected terminal nucleotide.
  • the at least one initiator comprise at least one cleavable group and there is a step (f) after step (e) comprising cleaving said at least one cleavable group, thereby releasing the polynucleotide from the surface.
  • the at least one cleavable group is enzymatically cleaved.
  • the step (f) of cleaving comprises treating said polynucleotide with an endonuclease activity.
  • the endonuclease activity is an endonuclease V or endonuclease VIII activity.
  • an enzymatic cleavage site comprises an inosine or a deoxyinosine, which can be cleaved by a eukaryotic or a prokaryotic endonuclease V enzyme, respectively.
  • an Escherichia coli endonuclease V is used for cleavage of an enzymatic cleavage site comprising a deoxyinosine.
  • the invention also covers a kit for enzymatically synthesizing a polynucleotide comprising at least one 3’-O-protected nucleoside triphosphate, a template-free polymerase and at least one salt compound at a concentration comprised between 500mM and 10 M.
  • the salt compound concentration is comprised between 1 M and 9 M, preferably between 2 M and 8 M, more preferably 3 M and 7 M.
  • the present invention encompasses the use of various salt compounds.
  • template-free polymerases such as TdT
  • TdT template-free polymerases
  • various salts which can be used in the present invention, e.g. magnesium salts, sodium salts or potassium salts, such as magnesium acetate, magnesium chloride, potassium acetate or potassium chloride.
  • the at least one salt compound comprises an alkali metal or alkaline earth metals salt, preferably an alkali metals salt, more preferably an alkali metals salt chosen among the group of Lithium and Sodium.
  • template-free polymerases such as TdT, are known to be active in the presence of various salts, which can be used in the present invention .
  • the said template-independent DNA polymerase is a terminal deoxynucleotidyl transferase (TdT).
  • the salt compound, the template-free polymerase and the 3’-O- protected nucleoside triphosphate are packaged in separate containers, e.g. as three separate solutions.
  • the kit also comprises a pH buffer with a pKa between 5 and 9.
  • the pH buffer is cacodylic acid.
  • An exemplary cleavable nucleotide may be a uracil nucleotide which is cleaved by uracil DNA glycosylase. If the elongated initiator polynucleotide does not contain a completed sequence, then the 3’-O- protection groups are removed to expose free 3’-hydroxyls (103) and the elongated initiator polynucleotides are subjected to another cycle of nucleotide addition and deprotection.
  • the terms “protected” and “blocked” in reference to specified groups are used interchangeably and are intended to mean a moiety is attached covalently to the specified group that prevents a chemical change to the group during a chemical or enzymatic process.
  • the prevented chemical change is a further, or subsequent, extension of the extended fragment (or “extension intermediate”) by an enzymatic coupling reaction.
  • an “initiator” (or equivalent terms, such as, “initiating fragment,” “initiator nucleic acid,” “initiator oligonucleotide,” or the like) usually refers to a short oligonucleotide sequence with a free 3 ’-hydroxyl at its end, which can be further elongated by a template-free polymerase, such as TdT.
  • the initiating fragment is a DNA initiating fragment.
  • the initiating fragment is an RNA initiating fragment.
  • an initiating fragment possesses between 3 and 100 nucleotides, in particular between 3 and 20 nucleotides.
  • the initiating fragment is single-stranded. In alternative embodiments, the initiating fragment may be double-stranded.
  • an initiator oligonucleotide may be attached to a synthesis support by its 5 ’end; and in other embodiments, an initiator oligonucleotide may be attached indirectly to a synthesis support by forming a duplex with a complementary oligonucleotide that is directly attached to the synthesis support, e.g. through a covalent bond.
  • a synthesis support is a solid support which may be a discrete region of a solid planar solid or may be a bead.
  • an initiator may comprise a non-nucleic acid compound having a free hydroxyl to which a TdT may couple a 3’-O-protected dNTP, e.g. Baiga, U.S. patent publications US2019/0078065 and US2019/0078126.
  • polynucleotides with the desired nucleotide sequence may be released from initiators and the solid supports by cleavage.
  • a wide variety of cleavable linkages or cleavable nucleotides may be used for this purpose.
  • cleaving the desired polynucleotide leaves a natural free 5 ’-hydroxyl on a cleaved strand; however, in alternative embodiments, a cleaving step may leave a moiety, e.g. a 5’-phosphate, that may be removed in a subsequent step, e.g. by phosphatase treatment.
  • an ordered sequence of nucleotides are coupled to an initiator nucleic acid using a template-free polymerase, such as TdT, in the presence of 3’- O-protected dNTPs in each synthesis step.
  • a template-free polymerase such as TdT
  • the method of synthesizing an oligonucleotide comprises the steps of (a) providing an initiator having a free 3’-hydroxyl (100); (b) reacting (104) under extension conditions the initiator or an extension intermediate having a free 3 ’-hydroxyl with a template-free polymerase in the presence of a 3 ’-0 -protected nucleoside triphosphate to produce a 3’-O-protected extension intermediate (106); (c) deprotecting the extension intermediate to produce an extension intermediate with a free 3 ’-hydroxyl (108); and (d) repeating steps (b) and (c) (110) until the polynucleotide is synthesized.
  • an initiator is provided as an oligonucleotide attached to a solid support, e.g. by its 5’ end.
  • the above method may also include washing step after each reaction, or extension, step, as well as after each de-protecting step.
  • the step of reacting may include a sub-step of removing unincorporated nucleoside triphosphates, e.g. by washing, after a predetermined incubation period, or reaction time. Such predetermined incubation periods or reaction times may be a few seconds, e.g. 30 sec, to several minutes, e.g. 30 min.
  • base protecting moieties for exocyclic amines are selected so that hydrogens of the protected nitrogen cannot participate in hydrogen bonding, thereby preventing the formation of such secondary structures. That is, base protecting moieties may be employed to prevent the formation of hydrogen bonds, such as are formed in normal base pairing, for example, between nucleosides A and T and between G and C.
  • the base protecting moieties may be removed and the polynucleotide product may be cleaved from the solid support, for example, by cleaving it from its initiator.
  • elongation reactions may be performed at higher temperatures using thermal stable template-free polymerases.
  • a thermal stable template-free polymerase having activity above 40°C may be employed; or, in some embodiments, a thermal stable template-free polymerase having activity in the range of from 40-85°C may be employed; or, in some embodiments, a thermal stable template-free polymerase having activity in the range of from 40-65 °C may be employed.
  • elongation conditions may include adding solvents to an elongation reaction mixture that inhibit hydrogen bonding or base stacking.
  • solvents include water miscible solvents with low dielectric constants, such as dimethyl sulfoxide (DMSO), methanol, and the like.
  • elongation conditions may include the provision of chaotropic agents that include, but are not limited to, n-butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, urea, and the like.
  • elongation conditions include the presence of a secondary-structure-suppressing amount of DMSO.
  • elongation conditions may include the provision of DNA binding proteins that inhibit the formation of secondary structures, wherein such proteins include, but are not limited to, singlestranded binding proteins, helicases, DNA glycolases, and the like.
  • 3’-O-blocked dNTPs without base protection may be purchased from commercial vendors or synthesized using published techniques, e.g. U.S. patent 7057026; Guo et al, Proc. Natl. Acad. Sci., 105(27): 9145-9150 (2008); Benner, U.S. patents 7544794 and 8212020; International patent publications W02004/005667, WO91/06678; Canard et al, Gene (cited herein); Metzker et al, Nucleic Acids Research, 22: 4259-4267 (1994); Meng et al, J. Org. Chem., 14: 3248-3252 (3006); U.S. patent publication 2005/037991. 3’-O-blocked dNTPs with base protection may be synthesized as described below.
  • the above method of Fig. 1 may further include a step (e) removing base protecting moieties, which in the case of acyl or amidine protection groups may (for example) include treating with concentrated ammonia.
  • the above method may also include one or more capping steps in addition to washing steps after the reacting, or extending, step.
  • a first capping step may cap, or render inert to further extensions, unreacted 3 ’-OH groups on partially synthesized polynucleotides.
  • Such capping step is usually implemented after a coupling step, and whenever a capping compound is used, it is selected to be unreactive with protection groups of the monomer just coupled to the growing strands.
  • capping steps may be implemented by coupling (for example, by a second enzymatic coupling step) a capping compound that renders the partially synthesized polynucleotide incapable of further couplings, e.g. with TdT.
  • Such capping compounds may be a dideoxynucleoside triphosphate.
  • non-extended strands with free 3’- hydroxyls may be degraded by treating them with a 3 ’-exonuclease activity, e.g. Exo I.
  • strands that fail to be deblocked may be treated to either remove the strand or render it inert to further extensions.
  • a second capping step may be implemented after a deprotection step, to render the affected strands inert from any subsequent coupling or deprotection any 3’-0 protection, or blocking groups.
  • Capping compounds of such second capping step are selected so that they do not react with free 3 ’-hydroxyls that may be present.
  • such second capping compound may be a conjugate of an aldehyde group and a hydrophobic group. The latter group permits separation based on hydrophobicity, e.g. Andrus, U.S. patent 5047524.
  • reaction conditions for an elongation step may comprising the following: 2.0 pM purified TdT; 125-600 pM 3’-O-blocked dNTP (e.g. 3’-O-NH2-blocked dNTP); about 10 to about 500 mM potassium cacodylate buffer (pH between 6.5 and 7.5) and from about 0.01 to about 10 mM of a divalent cation (e.g. C0CI2 or MnCl 2), where the elongation reaction may be carried out in a 50 pL reaction volume, at a temperature within the range RT to 45°C, for 3 minutes.
  • a divalent cation e.g. C0CI2 or MnCl 2
  • reaction conditions for a deblocking step may comprise the following: 700 mM NaNCh; 1 M sodium acetate (adjusted with acetic acid to pH in the range of 4.8-6.5), where the deblocking reaction may be carried out in a 50 pL volume, at a temperature within the range of RT to 45°C for 30 seconds to several minutes. Washes may be performed with the cacodylate buffer without the components of the coupling reaction (e.g. enzyme, monomer, divalent cations).
  • the coupling reaction e.g. enzyme, monomer, divalent cations
  • the steps of deblocking and/or cleaving may include a variety of chemical or physical conditions, e.g. light, heat, pH, presence of specific reagents, such as enzymes, which are able to cleave a specified chemical bond.
  • Guidance in selecting 3’- O-blocking groups and corresponding de-blocking conditions may be found in the following references, which are incorporated by reference: Benner, U.S. patents 7544794 and 8212020; U.S. patent 5808045; U.S. patent 8808988; International patent publication WO91/06678; and references cited below.
  • the cleaving agent (also sometimes referred to as a de-blocking reagent or agent) is a chemical cleaving agent, such as, for example, dithiothreitol (DTT).
  • a cleaving agent may be an enzymatic cleaving agent, such as, for example, a phosphatase, which may cleave a 3 ’-phosphate blocking group. It will be understood by the person skilled in the art that the selection of deblocking agent depends on the type of 3 ’-nucleotide blocking group used, whether one or multiple blocking groups are being used, whether initiators are attached to living cells or organisms or to solid supports, and the like, that necessitate mild treatment.
  • kits for carrying out methods of the invention includes kits for carrying out methods of the invention.
  • a kit for synthesizing a polynucleotide comprises at least one 3-O-NH2-alkyne-nucleoside triphosphate.
  • such 3-O-NH2-alkyne-nucleoside triphosphate includes a 5- octa(l,7)diynyl- 3'aminoxy-dUTP.
  • kits of the invention may further comprise an initiator attached to a support by a 5’ end and having a deoxyinosine penultimate to a 3’ end and free 3’- hydroxyl.
  • a kit of the invention further includes an endonuclease V capable of cleaving an initiator-polynucleotide conjugate 3’ of a terminal nucleotide of the initiator.
  • the endonuclease V has a capture moiety to permit removal from a reaction mixture.
  • capture moiety is a His tag.
  • initiators of a kit have a 3 ’-terminal sequence of 5’-dI-dT-3’. In some embodiments, initiators of a kit have a 3’- terminal sequence of 5’-dI-dG-3’. In some embodiments, initiators of a kit have a 3 ’-terminal sequence of 5’-dI-dA-3’. In some embodiments, initiators of a kit have a 3 ’-terminal sequence of 5’-dI-dT-3’, 5’-dI-dG-3’, or 5’-dI-dA-3’. In some embodiments, such support is a solid support.
  • kits of the invention may further include a template-free polymerase and 3’-O-blocked nucleoside triphosphates of one or more of deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine.
  • template-free polymerase may be a TdT.
  • TdT may be a TdT variant described herein.
  • a kit of the invention may further include a de-blocking agent which is capable of removing 3’ blocking groups from incorporated 3’-O-blocked nucleotides.
  • kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials. Such contents may be delivered to the intended recipient together or separately.
  • a first container may contain an enzyme for use in an assay, while a second or more containers contain mutually quenching fluorescent labels and/or quenching agents.
  • reaction site refers to a localized region where at least one of the designated reactions for enzymatic synthesis of a polynucleotide may occur.
  • a reaction site may include support surfaces of a reaction structure or substrate where a substance may be immobilized thereon.
  • the reaction site may be a discrete region of a region of space where a discrete group of polynucleotide strands are synthesized.
  • the reaction site may be a discrete region of a solid support, such as a planar solid, or may be a bead where a discrete group of polynucleotide strands are synthesized.
  • a compound added at the reaction site or in the reaction site is therefore added at the localized region where at least one of the designated reactions for enzymatic synthesis of a polynucleotide may occur.
  • Polynucleotide or “oligonucleotide” are used interchangeably and each mean a linear polymer of nucleotide monomers or analogs thereof.
  • Monomers making up polynucleotides and oligonucleotides are capable of specifically binding to a natural polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like.
  • Such monomers and their internucleosidic linkages may be naturally occurring or may be analogs thereof, e.g. naturally occurring or non-naturally occurring analogs.
  • Non-naturally occurring analogs may include PNAs, phosphorothioate internucleosidic linkages, bases containing linking groups permitting the attachment of labels, such as fluorophores, or haptens, and the like.
  • PNAs phosphorothioate internucleosidic linkages
  • bases containing linking groups permitting the attachment of labels such as fluorophores, or haptens, and the like.
  • labels such as fluorophores, or haptens, and the like.
  • oligonucleotide or polynucleotide requires enzymatic processing, such as extension by a polymerase, ligation by a ligase, or the like, one of ordinary skill would understand that oligonucleotides or polynucleotides in those instances would not contain certain analogs of internucleosidic linkages, sugar moieties, or bases at any or some positions.
  • Polynucleotides typically range in size from a few monomeric units
  • oligonucleotides when they are usually referred to as “oligonucleotides,” to several thousand monomeric units.
  • ATGCCTG a sequence of letters (upper or lower case), such as "ATGCCTG”
  • A denotes deoxyadenosine
  • C denotes deoxycytidine
  • G denotes deoxyguanosine
  • T denotes thymidine
  • I denotes deoxyinosine
  • U denotes uridine, unless otherwise indicated or obvious from context.
  • polynucleotides comprise the four natural nucleosides (e.g. deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA or their ribose counterparts for RNA) linked by phosphodiester linkages; however, they may also comprise non-natural nucleotide analogs, e.g. including modified bases, sugars, or internucleosidic linkages.
  • nucleosides e.g. deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA or their ribose counterparts for RNA
  • non-natural nucleotide analogs e.g. including modified bases, sugars, or internucleosidic linkages.
  • oligonucleotide or polynucleotide substrate requirements for activity e.g. single stranded DNA, RNA/DNA duplex, or the like
  • selection of appropriate composition for the oligonucleotide or polynucleotide substrates is well within the knowledge of one of ordinary skill, especially with guidance from treatises, such as Sambrook et al, Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory, New York, 1989), and like references.
  • the oligonucleotide and polynucleotide may refer to either a single stranded form or a double stranded form (i.e. duplexes of an oligonucleotide or polynucleotide and its respective complement). It will be clear to one of ordinary skill which form or whether both forms are intended from the context of the terms usage.
  • Primer means an oligonucleotide, either natural or synthetic that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3’ end along the template so that an extended duplex is formed.
  • Extension of a primer is usually carried out with a nucleic acid polymerase, such as a DNA or RNA polymerase.
  • the sequence of nucleotides added in the extension process is determined by the sequence of the template polynucleotide.
  • primers are extended by a DNA polymerase.
  • Primers usually have a length in the range of from 14 to 40 nucleotides, or in the range of from 18 to 36 nucleotides. Primers are employed in a variety of nucleic amplification reactions, for example, linear amplification reactions using a single primer, or polymerase chain reactions, employing two or more primers. Guidance for selecting the lengths and sequences of primers for particular applications is well known to those of ordinary skill in the art, as evidenced by the following references that are incorporated by reference: Dieffenbach, editor, PCR Primer: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Press, New York, 2003).
  • the “minimum free energy (MFE)” of a sequence or sequences is the minimum value among free energies of all possible secondary structures of a sequence (or sequences). It is well known that a secondary structure with a small Gibbs standard free energy is more stable than the ones with larger Gibbs standard free energies. So, the Gibbs standard free energy values can be used to measure the quality of DNA sequences (Kawashimo, S., Ono, H., Sadakane, K., Yamashita, M. (2008). Dynamic Neighborhood Searches for Thermodynamically Designing DNA Sequence. In: Garzon, M.H., Yan, H. (eds) DNA Computing. DNA 2007. Lecture Notes in Computer Science, vol 4848. Springer, Berlin, Heidelberg).
  • the “standard Gibbs energy of formation” of a compound is the change of Gibbs energy resulting from the formation of 1 mole of the compound from its elemental components under standard state conditions.
  • Hard codons are defined as codons that are hard to add to the polynucleotide during elongation. It is especially true for the last nucleotide of the codon.
  • Exemplary hard codons are shown in Table 1.
  • Elongation time is the time period during which the template-free polymerase has elongation activity, in the presence of the elongation substrates, namely a nucleic acid initiator and one or more 3 ’ -O-protected nucleoside triphosphates.
  • the elongation typically starts from the addition of the 3 ’ -O-protected nucleoside triphosphate and polymerase at the reaction site and ends when the unreacted 3’-O- protected nucleoside triphosphates and the polymerase are removed from the reaction site by washing.
  • “Purity” is defined as the ratio between the number of synthesized polynucleotides obtained with the desired sequence and the total number of synthesized polynucleotides. [0080] In “at least one polynucleotide,” the term “one polynucleotide” is considered to be one nucleotide sequence of a polynucleotide but more than one molecule of this polynucleotide of the same sequence can be present.
  • a first set of tests were conducted using sequences Nl, N2, N4 (SEQ ID NO 3) and N6 (SEQ ID NO 4).
  • the different concentrations of NaCl can be selected from 0, 50, 100 and 200 mM and the addition time can be independently selected from 0, 50, 121 and 192 seconds.
  • the total elongation time is 240 sec.
  • a control called STD was performed with no NaCl addition at a buffer concentration of 500 mM of cacodylic acid.
  • the buffer concentration was set at a concentration of 200 mM or 500 mM of cacodylic acid.
  • a lower buffer concentration was used for a second set of tests with sequences Nl, N2, N8 (SEQ ID NO 5) and N9.
  • the conditions used are listed on Table 2.
  • the buffer concentration was set at a concentration of 100 mM or 200 mM of cacodylic acid.
  • the NaCl concentration used is 200 mM or 300 mM.

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Abstract

La présente invention concerne des procédés et des kits pour la synthèse enzymatique de polynucléotides sans gabarit avec une étape consistant à ajouter des composés salins pendant l'élongation du polynucléotide.
PCT/EP2024/066486 2023-06-15 2024-06-13 Addition de sels pendant la synthèse enzymatique de polynucléotides Pending WO2024256604A1 (fr)

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