Template-independent stepwise de novo synthesis of long polynucleotides

Abstract

This disclosure provides, in particular, a method for synthesizing long polynucleotides, and for example, an improved polynucleotide synthesis method that can achieve high step yields and / or low error rates for long polynucleotides, for example, using a stepwise template-independent de novo synthesis method.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits and priority of U.S. Provisional Patent Application No. 63 / 509,522, filed June 21, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing electronically filed in XML format, which is incorporated herein by reference in its entirety. The XML file, created on June 14, 2024, is named ABB-013WO_SL.xml and has a size of 99.9 kilobytes. [Background technology]

[0003] background Polynucleotide synthesis generates nucleotide chains, the building blocks of DNA and RNA. Current standard de novo DNA synthesis is based on the nucleoside phosphoramidite method (commonly referred to as "chemical synthesis"), which synthesizes the desired sequence by stepwise linking protected monomers. Such reactions are carried out in organic solvents using highly reactive activated monomers, and such conditions cause side reactions that damage the elongating chain, limiting the yield of the full-length product. The resulting impurities are difficult or impossible to separate from the desired oligonucleotide product, thus limiting the usefulness of methods for producing sequences longer than approximately 200 bases.

[0004] Alternatively, various enzymatic de novo DNA synthesis strategies using template-independent polymerases have recently been developed, enabling environmentally friendly synthesis of longer DNA molecules than chemical synthesis. However, step yields and lengths have not yet improved significantly. In some cases, step yields may begin to decline once the synthesized polynucleotide reaches a certain length. For polynucleotides up to 500 or 1000 nucleotides in length, even if step yields are stable, very high step yields are required to obtain a reasonable number of fully synthesized long polynucleotides by stepwise de novo synthesis.

[0005] Therefore, there is a need for improved polynucleotide synthesis methods that achieve high step yields and / or low error rates and / or long polynucleotide synthesis times through template-independent, stepwise de novo synthesis. [Overview of the project]

[0006] overview In particular, this disclosure provides technologies (e.g., methods for synthesizing polynucleotides) that bring about advances and improvements in the synthesis of long polynucleotides. In part, this disclosure is based on methods for synthesizing long polynucleotides, such as improved methods that not only yield higher step yields and / or lower error rates than conventional methods, but also produce long polynucleotides (e.g., about 500 to about 1000 nucleotides or longer) well.

[0007] In some embodiments, the present disclosure provides a template-independent de novo synthesis method for long single-stranded polynucleotides, the synthesis method being: To provide a substrate, wherein the substrate contains a plurality of free hydroxyl groups that are bonded to the substrate and are suitable for nucleotide bonding, The nucleotides attached to the protecting group are attached to the plurality of free hydroxyl groups, Removing the protecting group from the bound nucleotide, To obtain multiple de novo-synthesized polynucleotides of at least 500 nucleotides in length, steps (b) and (c) are repeated according to a predetermined nucleotide sequence (e.g., a reference sequence), This includes, where the error rate observed for each bond is less than 1% compared to the predetermined nucleotide sequence.

[0008] In some embodiments, the de novo-synthesized long polynucleotide is at least 600 nucleotides long, at least 700 nucleotides long, at least 800 nucleotides long, at least 900 nucleotides long, at least 1000 nucleotides long, at least 1100 nucleotides long, at least 1200 nucleotides long, at least 1300 nucleotides long, at least 1400 nucleotides long, at least 1500 nucleotides long, at least 1600 nucleotides long, at least 1700 nucleotides long, at least 1800 nucleotides long, at least 1900 nucleotides long, or at least 2000 nucleotides long.

[0009] In some embodiments, the de novo-synthesized long polynucleotides are 500-1000 nucleotides long, 500-1500 nucleotides long, 500-2000 nucleotides long, or 500-2500 nucleotides long.

[0010] In some embodiments, the error rates observed for each coupling are 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.

[0011] In some embodiments, the bonding 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.

[0012] In some embodiments, template-independent polynucleotide synthesis for obtaining 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.

[0013] In some embodiments, template-independent polynucleotide synthesis is performed at rates of 5 to 25 nucleotides per hour, 10 to 30 nucleotides per hour, 15 to 45 nucleotides per hour, or 20 to 60 nucleotides per hour.

[0014] In some embodiments, the multiple de novo-synthesized polynucleotides on the substrate include 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, and at least 10 × 10 5 A number of polynucleotides, at least 10 × 10 6 A number of polynucleotides, at least 10 × 10 7 A number of polynucleotides, at least 10 × 10 8 A number of polynucleotides, at least 10 × 10 9 A number of polynucleotides, at least 10 × 10 10 A number of polynucleotides, at least 10 × 10 11individual polynucleotides, at least 10×10 12 individual polynucleotides, at least 10×10 13 individual polynucleotides, or at least 10×10 14 individual polynucleotides.

[0015] In some embodiments, the free hydroxyl group is at the end of a plurality of starting oligonucleotides or extended polynucleotides attached to the substrate.

[0016] In some embodiments, the starting oligonucleotide contains a single-stranded region at the 3' end.

[0017] In some embodiments, the starting oligonucleotide is hybridized to an oligonucleotide attached to the substrate.

[0018] In some embodiments, the starting oligonucleotide is covalently attached to the substrate.

[0019] In some embodiments, the nucleotide binding is carried out by an enzyme.

[0020] In some embodiments, the nucleotide binding is carried out by the catalytic action of a polymerase.

[0021] In some embodiments, the polymerase is a template-independent polymerase.

[0022] In some embodiments, the template-independent polymerase is covalently attached to the nucleotide.

[0023] In some embodiments, the template-independent polymerase is terminal deoxynucleotidyl transferase (TdT), or a variant thereof.

[0024] In some embodiments, the polymerase is an RNA polymerase.

[0025] In some embodiments, the protecting group is a template-independent polymerase bound to a nucleotide.

[0026] In some embodiments, the protecting group includes a linker to be cleaved, which binds the nucleotide to a template-independent polymerase.

[0027] In some embodiments, the protecting group is a 3'-O-protecting group.

[0028] In some embodiments, removing a protecting group involves removing a 3'-O-protecting group from a nucleotide in order to leave a free 3'-hydroxyl group.

[0029] In some embodiments, the protecting group is a 2' or 3' modification of the nucleotide.

[0030] In some embodiments, the 2' modification is selected from the group consisting of -H, -OH, -F, -OMe, -N3, -NH2, and -Ara.

[0031] In some embodiments, the 3' modification is selected from the group consisting of -H, -OH, -OCH2N3, -ONH2, and -O allyl.

[0032] In some embodiments, the protecting group is a reversible terminator.

[0033] In some embodiments, a given 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%.

[0034] In some embodiments, a given 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%.

[0035] In some embodiments, the binding occurs in the presence of a phosphatase. In some embodiments, the phosphatase is an inorganic pyrophosphatase.

[0036] In some embodiments, the nucleotides bound to the protecting group are nucleotide-polymerase complexes, and the complexes are treated with phosphatases.

[0037] In some embodiments, bonding occurs in the presence of a divalent cation, and the total concentration of the divalent cation present in the bonding reaction volume is approximately 500 μM or less.

[0038] In some embodiments, the total concentration of divalent cations present in the reaction volume is approximately 250 μM or less, approximately 125 μM or less, or approximately 50 μM or less.

[0039] In some embodiments, the divalent cation present at the highest concentration in the reaction volume is cobalt (Co2+) or zinc (Zn2+).

[0040] In some embodiments, at least one divalent cation is selected from Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Fe2+, Ni2+, Cu2+, and Zn2+, or a combination thereof.

[0041] In some embodiments, the binding reaction is carried out in the absence of Mg2+.

[0042] In some embodiments, the nucleotide comprises one or more modifications to the hydrogen-bond-forming N or O on the nucleobase.

[0043] In some embodiments, the attached nucleotide comprises one or more alkylated nucleobases after removal of the protecting group. In some such embodiments, the method further comprises contacting the de novo synthesized polynucleotide with an alkyltransferase.

[0044] In some embodiments, the alkyltransferase belongs to EC 2.1.1.63.

[0045] In some embodiments, the alkyltransferase is selected from the alkyltransferases described in Table 1 or Table 2.

[0046] In some embodiments, the alkyltransferase is O6-alkylguanine DNA alkyltransferase.

[0047] In some embodiments, the alkyltransferase is AlkB.

[0048] In some embodiments, the alkylated nucleobase is It is represented by TIFF2026522421000002.tif42165, where X is -C(R2)= or -N=, R1 is selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, C1-6 alkynyl, and -(CH2)0-3Ph, R1 may be substituted with 1 to 6 R1a, each R1a is independently selected from halogen, C1-6 alkyl, -(CH2)0-3OR1b, -NO2, -N3, -OPO2OH, and -(CH2)0-3NHR1b, each R1b is independently selected from hydrogen, C1-6 alkyl, -C(O)(C1-6 alkyl), C1-6 haloalkyl, -C(O)(C1-6 haloalkyl), and -CH2OAc, R2 is selected from the group consisting of hydrogen and an optionally substituted C1-4 alkyl chain, where 1 to 2 methylene units may be independently replaced by -O-, -N(Ra)-, -C(O)-, -S-, -S(O)-, -S(O)2-, or phenylene, R2 may be substituted with 1 to 6 R2a, each R2a is independently selected from halogen, C1-6 alkyl, -(CH2)0-3OR2b, -NO2, -N3, -OPO2OH, and -(CH2)0-3NHR2b, and each R2b is independently selected from hydrogen and C1-6 alkyl.

[0049] In some embodiments, R1 is C1-4 alkyl.

[0050] In some embodiments, R1 is selected from methyl, ethyl, n-propyl, and n-butyl.

[0051] In some embodiments, R1 is selected from the group consisting of TIFF2026522421000003.tif53165.

[0052] In some embodiments, R1a is -OR1b.

[0053] In some embodiments, R1b is hydrogen.

[0054] In some embodiments, the alkylated nucleic acid base of the polynucleotide is The selection is made from the group consisting of TIFF2026522421000004.tif91165, TIFF2026522421000005.tif220165, and TIFF2026522421000006.tif63165.

[0055] In some embodiments, the bound nucleotide, after removal of the protecting group, includes one or more modifications to the base-pairing nitrogen or oxygen on the nucleic acid base.

[0056] In some embodiments, the bound nucleotides are as follows (B): TIFF2026522421000007.tif37165(B), Represented by the formula, where R is ribose polyphosphate or deoxyribose polyphosphate, Y is a nucleic acid base, L-R1 is a protecting group, L is bonded to a base-pairing nitrogen or oxygen of the nucleic acid base, and R1 is selected from the group consisting of hydrogen, -OH, -N(Rb)2, and -SH, where each Rb is independently hydrogen or an optionally substituted C1-6 alkyl group.

[0057] In some embodiments, L is -Z-L1-L2-, and Z is selected from the group consisting of bond, -C(O)-, -C(O)CH2-, -C(O)C(RL)2-, -C(O)CH(RL)-, -C(O)O-, and -C(O)N(H)-.

[0058] L1 is a bond, Selected from the group consisting of TIFF2026522421000008.tif47165, L1 may be substituted with 1 to 4 RLs, each RL independently selected from the group consisting of halogens, hydroxyls, oxos, and optionally substituted C1-C3 alkyls, where two R1s may form a 3 to 6-membered carbocyryl ring together with intervening atoms(s), L2 is selected from the group consisting of bonded or substituted C1-12 alkylene chains, C4-C20 polyethylene glycols, optionally substituted C2-12 alkenylene chains, and optionally substituted C2-12 alkylylene chains, where one to six methylene units may independently be replaced with -O-, -N(Rb)-, -C(O)-, -S-, -S(O)-, -S(O)2-, or phenylene. W is selected from the group consisting of -O-, -S-, -S(O)2-, and -N(Rb)-, each Ra is a halogen, -Me, or -OMe, and each Rb is independently hydrogen or C1-6 alkyl. n is either 1 or 2, and L1 and Z cannot be joined at the same time.

[0059] In some embodiments, Z is a bond when L is bonded to a base-pairing oxygen of a nucleic acid base.

[0060] In some embodiments, when L is bonded to the base-pairing nitrogen of a nucleic acid base, 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)-.

[0061] In some embodiments, L1 is The filename is TIFF2026522421000009.tif17165, where L1 may be replaced by 1 to 4 RLs, n is 1 or 2, and W is selected from the group consisting of -O-, -S-, -S(O)2-, and -N(Rb)-.

[0062] In some embodiments, Selected from the group consisting of TIFF2026522421000010.tif42165, L1 may be substituted with 1 to 4 RLs.

[0063] In some embodiments, each RL is independently optionally substituted C1-C3 alkyl, where two R1s may together with intervening atom(s) form a 3- to 6-membered carbocyclic ring.

[0064] In some embodiments, RL is optionally substituted methyl.

[0065] In some embodiments, L1 is Selected from the group consisting of TIFF2026522421000011.tif63165.

[0066] In some embodiments, Z is -C(O)O-.

[0067] In some embodiments, Z is -C(O)N(H)-.

[0068] In some embodiments, Z is a bond.

[0069] In some embodiments, Z is -C(O)-.

[0070] In some embodiments, -Z-L1-L2-R1 is Selected from the group consisting of TIFF2026522421000012.tif68165.

[0071] In some embodiments, L2 is an optionally substituted C1-12 alkylene chain, where 1 to 6 methylene units may independently be replaced by -O-, -N(Rb)-, -C(O)-, -S-, -S(O)-, -S(O)2-, or phenylene.

[0072] In some embodiments, L2 is a substituted C1-12 alkylene chain, where 1 to 6 methylene units may be independently replaced by -O-.

[0073] In some embodiments, L2 is a substituted C2-6 alkylene chain, where 1 to 3 methylene units may be independently replaced by -O-.

[0074] In some embodiments, -Z-L1-L2-R1 is Selected from the group consisting of TIFF2026522421000013.tif68165.

[0075] In some embodiments, binding involves immersing the substrate in a solution containing nucleotides and a template-independent polymerase.

[0076] In some embodiments, the protecting group is a polymerase, which is bound to the nucleotide via a cleavable linker.

[0077] In some embodiments, the cleavable linker contains an amino acid ester.

[0078] In some embodiments, the amino acid ester is bonded to an amino acid.

[0079] In some embodiments, the amine group of the amino acid ester is bonded to the amino acid.

[0080] In some embodiments, the cleavable linker comprises a peptide of at least two, at least three, at least four, or at least five amino acids bonded to the amine group of the amino acid ester.

[0081] In some embodiments, one or more amino acids are 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.

[0082] In some embodiments, the amino acid is glycine, or the multiple amino acids include glycine.

[0083] In some embodiments, the amino acids are amino acids that do not exist in nature, or the amino acids include amino acids that do not exist in nature.

[0084] In some embodiments, the cleavable linker is bound to the alpha-phosphate, sugar, or nucleic acid base of the nucleotide.

[0085] In some embodiments, amino acid esters are Represented by TIFF2026522421000014.tif27165, in which R1 and R1' are each independently selected from hydrogen and / or substituted C1-6 alkyl groups, or together with the atoms to which they are bonded, they may form / or substituted C3-C7 carbocyclic rings.

[0086] In some embodiments, amino acid esters are The compound is selected from the group consisting of TIFF2026522421000015.tif32165.

[0087] In some embodiments, the linker has the following structure: The formula includes TIFF2026522421000016.tif32165, where R1 and R1' are each independently selected from hydrogen and / or optionally substituted C1-6 alkyl groups, or together with the atom to which they are bonded, may 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 groups, phenyl groups, C1-C6 carbocyclic rings, and 3-7 membered heterocycles; each R3 is hydrogen or an optionally substituted C1-6 alkyl group; and n is 1, 2, 3, 4, or 5.

[0088] In some embodiments, R3 is hydrogen.

[0089] In some embodiments, R2 is hydrogen.

[0090] In some embodiments, R2 is hydrogen, -Me, -isopropyl, -sec-butyl, isobutyl, -CH2Ph, -CH2OH, -CH2SH, -CH2CH2SCH3, -CH2COOH, -CH2CH2COOH, -CH2CONH2, -CH2CH2CONH2, -CH2CH2, -CH2CH2NH2, Selected from the group consisting of TIFF2026522421000017.tif47165.

[0091] In some embodiments, n is 1.

[0092] In some embodiments, R1 and R1' together form a C3-C7 carboncyclic ring, which may be substituted.

[0093] In some embodiments, R1 and R1' together form a C3 carboncyclic ring, which may be substituted.

[0094] In some embodiments, the linker is Includes the structure of TIFF2026522421000018.tif32165.

[0095] In some embodiments, the nucleotide bound to polymerase comprises the structure:Nuc-L1-L2-L3-Pol, where Nuc is a nucleotide, Pol is a polymerase, L1 is the first part of a linker that links the nucleotide to L2, and L2 is... The second part of the linker represented by TIFF2026522421000019.tif37165, where R1 and R1' are each independently selected from optionally substituted C1-6 alkyl groups, halogens, or together with the atom to which they are bonded, may form optionally substituted C3-C7 carbocyclic rings; each R2 is an optionally substituted group independently selected from the group consisting of hydrogen, C1-6 alkyl groups, phenyl groups, C1-C6 carbocyclic rings, and 3- to 7-membered heterocycles; each R3 is hydrogen or an optionally substituted C1-6 alkyl group; n is 0, 1, 2, 3, 4, or 5, where * indicates a bond site from L2 to L1, ** indicates a bond site from L2 to L3, L2 is cleavable, and L3 is a linker that links pol to L2.

[0096] In some embodiments, L1 is selected from the group consisting of a bonded, possibly substituted C1-12 alkylene chain, a C4-C20 polyethylene glycol, a possibly substituted C2-12 alkenylene chain, and a C2-12 alkylylene chain, where 1 to 6 methylene units of L1 may be independently replaced with -O-, -N(Rb)-, -N=C(H)-, -C(O)-, -S-, -S(O)-, -S(O)2-, possibly substituted phenylene, or possibly substituted cyclopropylene.

[0097] In some embodiments, L1 is The set includes TIFF2026522421000020.tif47165, and each Ra is independently selected from the group consisting of halogens, hydroxyls, cyanos, optionally substituted C1-6 alkyls, and optionally substituted C1-6 alkoxys.

[0098] In some embodiments, L2 is It contains an amino acid ester selected from the group consisting of TIFF2026522421000021.tif53165.

[0099] In some embodiments, L2 is Represented by TIFF2026522421000022.tif32165.

[0100] In some embodiments, L1 is bound to a nucleic acid base of a nucleotide.

[0101] In some embodiments, L1 is bonded to the nucleic acid base with oxygen or nitrogen involved in base pairing.

[0102] In some embodiments, nucleic acid bases are Selected from the group consisting of TIFF2026522421000023.tif99165.

[0103] In some embodiments, L1 is bound to the sugar of the nucleotide.

[0104] In some embodiments, L1 is bonded to the phosphate group of the nucleotide.

[0105] In some embodiments, the phosphoric acid is alpha-phosphate.

[0106] In some embodiments, the nucleotide is ribonucleotide polyphosphate or deoxyribonucleotide polyphosphate.

[0107] In some embodiments, the nucleotide is selected from the group consisting of adenine, guanine, cytosine, uracil, and thymine.

[0108] In some embodiments, the polymerase is a template-independent polymerase.

[0109] In some embodiments, the polymerase is TdT.

[0110] In some embodiments, the linker can be cleaved by a protease having esterase activity.

[0111] In some embodiments, the linker is cleavable by proteinase K.

[0112] In some embodiments, the linker can be cleaved at the ester group on L2, leaving behind a compound represented by Nuc-L1-OH after cleavage.

[0113] In some embodiments, the Disclosure provides a substrate having a plurality of linked polynucleotides, each at least 500 nucleotides in length, wherein the plurality of polynucleotides are sequences produced by stepwise template-independent polynucleotide synthesis, with an error rate of less than 1% per cycle compared to a given nucleotide sequence.

[0114] In some embodiments, the polynucleotide is at least 600 nucleotides long, at least 700 nucleotides long, at least 800 nucleotides long, at least 900 nucleotides long, at least 1000 nucleotides long, at least 1100 nucleotides long, at least 1200 nucleotides long, at least 1300 nucleotides long, at least 1400 nucleotides long, at least 1500 nucleotides long, at least 1600 nucleotides long, at least 1700 nucleotides long, at least 1800 nucleotides long, at least 1900 nucleotides long, or at least 2000 nucleotides long.

[0115] In some embodiments, the polynucleotides are 500-1000 nucleotides long, 500-1500 nucleotides long, 500-2000 nucleotides long, or 500-2500 nucleotides long.

[0116] In some embodiments, the acceptable error rates are 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, compared to a given sequence.

[0117] In some embodiments, the acceptable 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 compared to a given sequence.

[0118] In some embodiments, the multiple bound polynucleotides on the substrate include 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, and at least 10 × 10 5 A number of polynucleotides, at least 10 × 10 6 A number of polynucleotides, at least 10 × 10 7 A number of polynucleotides, at least 10 × 10 8 A number of polynucleotides, at least 10 × 10 9 A number of polynucleotides, at least 10 × 10 10 A number of polynucleotides, at least 10 × 10 11 A number of polynucleotides, at least 10 × 10 12 A number of polynucleotides, at least 10 × 10 13 10 polynucleotides, or at least 10 × 10 14 It contains [number] polynucleotides.

[0119] In some embodiments, the polynucleotide comprises one or more nucleotides having modifications to hydrogen-bonding N or O on the nucleic acid base. [Brief explanation of the drawing]

[0120] The aforementioned and other purposes, features, and advantages will become apparent from the following description of specific embodiments of this disclosure. Specific embodiments are also illustrated in the accompanying drawings, where the same reference numerals indicate the same parts in different drawings. The drawings are not necessarily to a fixed scale and instead focus on illustrating the principles of the various embodiments of this disclosure.

[0121] [Figure 1A] This figure shows a scheme for two-step repeated nucleic acid synthesis using a polymerase-nucleotide complex. In the first step, the complex binds to a DNA molecule via its linked dNTP moiety. In the second step, the bond between the polymerase and the elongated DNA molecule containing the dNTP moiety is cleaved, and the ends of the DNA molecule are deprotected for subsequent elongation. [Figure 1B] This figure shows a scheme for two-step repeated nucleic acid synthesis using a TdT-dNTP complex. The TdT-dNTP complex contains a TdT molecule site-specifically linked to a dNTP via a cleavable linker. [Figure 2] Figures (i) and (ii) below show: (i) typical enzymatic DNA synthesis using a free nucleotide having a 3' protecting group and an enzyme (which may be inhibited by the formation of secondary structures during synthesis); and (ii) synthesis using an improved complex provided herein, comprising using a polymerase-nucleotide complex containing a polymerase linked to a base-pairing N or O atom of a nucleic acid base. When the polymerase is cleaved from the nucleotide in each cycle, a nucleotide with a residual group, including part of the linker (residual group) at the N or O atom, is retained in the polynucleotide, which inhibits secondary structure formation. The residual group(s) can then be removed to produce a polynucleotide without residual groups. [Figure 3] This figure shows a scheme for nucleic acid synthesis using a TdT-dNTP complex. Here, the dNTP has an O-alkyl modification separate from its linker binding site. In repeated nucleic acid synthesis, the linker is cleaved using a cleavage agent that leaves residual groups, and the TdT is removed from the added dNTP. Once nucleic acid synthesis is complete, the O-alkyl group is removed from the nucleotide(s) using an AGT. [Figure 4] This figure shows a scheme for nucleic acid synthesis using a TdT-dNTP complex. Here, a cleaving agent is used to cleave the linker and remove TdT from the attached dNTP, resulting in a nucleotide containing the O-alkyl residue derived from the cleavage. Once nucleic acid synthesis is complete, an AGT is used to remove the O-alkyl group from the nucleotide(s). [Figure 5A] This figure shows an exemplary intramolecular cyclization reaction for removing residual or protecting groups. The residual or protecting groups include unsubstituted alkyl groups and substituted alkyl groups, where the alkyl group is bonded to an amide group bonded to the nitrogen on a nucleic acid base. [Figure 5B] This figure shows an exemplary intramolecular cyclization reaction for removing residual or protecting groups. The residual or protecting groups include unsubstituted alkyl groups and substituted alkyl groups, where the alkyl group is bonded to a carbamate group bonded to the nitrogen on a nucleic acid base. [Figure 6A] This figure shows an example of unshielded nucleotides that may be present in a polymerase-nucleotide complex reagent. [Figure 6B] This figure shows exemplary unshielded nucleotides that may be present in polymerase-nucleotide complex reagents. Figure 6B shows an exemplary unfolded, template-independent polymerase with exemplary tethered nucleotides (e.g., dNTPs). [Figure 6C]This figure shows exemplary unshielded nucleotides that may be present in a polymerase-nucleotide complex reagent. Figure 6A shows an exemplary template-independent polymerase with an exemplary unshielded nucleotide (e.g., deoxynucleoside triphosphate or "dNTP") tethered in the wrong position. Figure 6C illustrates exemplary "free" (or untethered) nucleotides (e.g., dNTPs) present in an exemplary complex reagent. In such a polymerase-nucleotide complex reagent, such free dNTPs may be present, for example, due to cleavage of the linker between the nucleotide and the polymerase (e.g., due to instability), or for example, due to incomplete removal of free nucleotides from the complex after complex synthesis. Each nucleotide in Figures 6A, 6B, and 6C has a 5' phosphate group that can be removed by phosphatase catalytic action. [Figure 6D] This figure shows an exemplary polymerase-nucleotide complex containing an exemplary shielded nucleotide (e.g., dNTP). In this figure, the exemplary nucleotide is tethered to the catalytic site of the folded polymerase, and the tethered polymerase sterically inhibits phosphatase cleavage at its 5' phosphate. [Figure 7] This block diagram shows a synthesis system suitable for an exemplary embodiment. [Figure 8] This figure shows a first exemplary configuration of the synthesis system. [Figure 9] This figure shows a second exemplary configuration of the synthesis system. [Figure 10] This figure shows exemplary members and exemplary wells suitable for exemplary embodiments. [Figure 11] This figure shows an exemplary cross-sectional shape of a member according to an exemplary embodiment. [Figure 12] This figure shows the longitudinal shape of an exemplary member according to an exemplary embodiment. [Figure 13A] This diagram shows the configuration of a floating member in an exemplary embodiment. [Figure 13B]This diagram shows the configuration of a floating member in an exemplary embodiment. [Figure 14] This figure shows an exemplary reaction plate used in an exemplary embodiment. [Figure 15] This figure shows a portion of an exemplary patterned surface used in an exemplary embodiment. [Figure 16] This is a flowchart showing exemplary steps that can be carried out in exemplary embodiments for synthesizing a polymer. [Figure 17] This figure shows exemplary operations that can be performed in an exemplary embodiment to carry out hybridization on the components of a component array. [Figure 18] This flowchart shows exemplary steps that can be carried out in exemplary embodiments as part of a synthesis process. [Figure 19] This flowchart shows exemplary steps that can be carried out in exemplary embodiments to implement a synthesis process cycle. [Figure 20] This figure shows an exemplary pattern in which a polymer extension solution is filled into the wells of a reaction plate in an exemplary embodiment. [Figure 21] This figure shows the results of the extension reaction. The extension reaction was carried out for 30 seconds, 1 minute, 2 minutes, 4 minutes, or 8 minutes using TdT and (i)dGTP (left column) and (ii)O6-methyldGTP (right column) as described in Example 1. The starting material was unmodified T35 oligonucleotide (SEQ ID NO: 48). The x-axis represents the approximate nucleotide length of the oligonucleotide, and the y-axis represents the relative fluorescence intensity of fluorescein at 517 nm. [Figure 22] This figure shows the results of the extension reaction. The extension reaction was carried out for 3.8 seconds, 6.6 seconds, 11.6 seconds, 19.2 seconds, 29 seconds, 45 seconds, 67 seconds, and 139 seconds, as described in Example 3, with nucleotides containing i) alkylated (left column) or ii) unmodified (right column) 6GC hairpins. [Figure 23]The figure shows the results of repeated nucleotide synthesis reactions using the TdT-dNTP complex as shown in Example 4. The TdT-dNTP complex uses i) a G nucleotide without alkylation at the O6 position (O7Et-G), ii) a G nucleotide with alkylation at the O6 position at only two locations (where the strongest secondary structure is predicted) (the rest of the G nucleotide is unalkylated) (O7Et-G and O6Bu-G), and iii) a G nucleotide with alkylation at the O6 position (O6Bu-G). [Figure 24] This figure shows an agarose gel visualized under UV light from the synthesized 50-base alkylated PCR amplification product. The synthesized 50-base alkylated product was treated with AGT for a predetermined time, as described in Example 5, or not. A non-alkylated positive control is also shown for reference. [Figure 25] This figure shows the results of repeated nucleotide synthesis reactions using a TdT-dNTP complex on a 50-base-length poly-G homopolymer (SEQ ID NO: 49). The synthesis reaction used G nucleotides with alkylated O6 positions, as described in Example 6. [Figure 26-1] This figure shows the results of repeated nucleotide synthesis reactions using a TdT-dNTP complex for a 40-nucleotide sequence expected to have a 15bp hairpin structure. The synthesis reaction used a G nucleotide with alkylated O6 position, as described in Example 7. Figure 26 discloses Sequence ID No. 35. [Figure 26-2] See the explanation in Figure 26-1. [Figure 27] This figure shows the reaction scheme, the detection of human AGT activity, and the gel. The activity of human AGT against various O6-alkylated G nucleotides in the synthesized polynucleotides was detected. The gel shows the results of the analysis for O6-methyl-G, O6-hydroxybutyl-G, O6-hydroxypropyl-G, N7-aminomethyl-O6-methyl-G, and O6-aminomethylbenzyl-G (as described in Example 8). [Figure 28A-1] This figure shows the results of the dealkylation reaction of oligonucleotides. [Figure 28A-2]Refer to the explanation in Figure 28A-1. [Figure 28B-1] This figure shows the results of the dealkylation reaction of oligonucleotides. The oligonucleotides contain a 3' G nucleotide with an O6-allyl-modified G nucleotide at the 3' end. The obtained oligonucleotides were measured by capillary electrophoresis. "Allyl-G" represents the control (untreated oligonucleotide with an O6-allyl-modified G nucleotide at the 3' end), and the other oligonucleotides were treated with the corresponding type of AGT as shown in Figures 28A and 28B. [Figure 28B-2] Refer to the explanation in Figure 28B-1. [Figure 29A] This figure shows the results of the poly-G synthesis reaction. [Figure 29B] This figure shows the results of the poly-G synthesis reaction. The poly-G synthesis reaction was carried out without O6 alkyl modification ("dGTP"), and also with O6 alkyl modification at the 3' end of the poly-T starting oligonucleotide (Figure 29A) and the 3' end of the poly-C starting oligonucleotide (Figure 29B). [Figure 30] This figure shows the incorporation of alkylated G nucleotides and alkylated U nucleotides at the 3' end of oligonucleotides by polymerase-nucleotide complexes, followed by polymerase cleavage and the retention of alkyl groups on the G nucleotide or U nucleotide. [Figure 31-1] Figure 30 shows the successful dealkylation of the synthesized oligonucleotide by AGT. Dealkylation leads to the synthesis of oligonucleotides without residual groups in the complex. [Figure 31-2] Figure 30 shows the successful dealkylation of the synthesized oligonucleotide by AGT. Dealkylation leads to the synthesis of oligonucleotides without residual groups in the complex. [Figure 32A] This is an HPLC chromatogram showing traces of photocleavable dGTP nucleotides (the starting material for photocleavage experiments). The X-axis represents minutes. The elution peak at 5.693 minutes indicates intact photocleavable dGTP nucleotides. [Figure 32B]This is an HPLC chromatogram showing traces of natural dGTP nucleotides (expected products of the photocleavage reaction). The X-axis represents minutes. The elution peak at 2.001 minutes indicates natural dGTP nucleotides. [Figure 32C] This is an HPLC chromatogram showing traces of the photocleavable dGTP reaction product after exposure to a 365 nm wavelength lamp for 120 minutes. The X-axis represents minutes. [Figure 33A] This is an HPLC chromatogram showing traces of photocleavable dATP nucleotides (the starting material for photocleavage experiments). The X-axis represents minutes. The elution peak at 5.693 minutes indicates intact photocleavable dATP nucleotides. [Figure 33B] This is an HPLC chromatogram showing traces of the photocleavable dATP reaction product after exposure to a 365 nm wavelength lamp for 120 minutes. The X-axis represents minutes. The elution peak at 3.426 minutes indicates native dATP nucleotides. [Figure 34A] This is a capillary electrophoresis ferrogram showing polynucleotide elongation products exposed to UV light after elongation with a complex containing modified dGTP and TdT having photocleavable nitrobenzyl groups. [Figure 34B] This is a capillary electrophoresis ferrogram showing polynucleotide elongation products exposed to UV light after elongation with a complex containing modified dGTP and TdT having photocleavable nitrobenzyl groups. [Figure 35] This is a capillary electrophoresis ferrogram showing polynucleotide elongation products by a complex having a linker bound to cytosine N4 and containing different cleavable residual groups. [Figure 36-1] This is a capillary electrophoresis ferrogram showing polynucleotide elongation products by a complex having a linker that binds to N6 of adenine and contains different cleavable residual groups. [Figure 36-2] Refer to the explanation in Figure 36-1. [Figure 37]This is a capillary electrophoresis ferrogram showing polynucleotide elongation products of complexes having linkers bonded to O4 of uracil and thymine and containing different cleavable residual groups. The X-axis represents the approximate oligonucleotide length by the number of nucleotides, and the Y-axis represents the relative fluorescence intensity. [Figure 38] This is a capillary electrophoresis ferrogram showing the polynucleotide elongation product of a complex having a linker bound to O6 of guanine and containing different cleavable residual groups. [Figure 39] This is a capillary electrophoresis ferrogram showing polynucleotide elongation products incubated at pH 7 or pH 8 for various durations after elongation with a complex containing modified dGTP and TdT having a detachable sulfone group. The first column contains control ssDNA. The control ssDNA serves as a marker for the migration location of a single native guanine at the 3' end of the starting oligonucleotide. Vertical dotted lines indicating the migration of native guanine and +1 sulfone-guanine are shown for clarity. [Figure 40] This is a capillary electrophoresis ferrogram showing the results of single-nucleotide elongation of the starting oligonucleotide. Single-nucleotide elongation was performed using dGTP with a sulfide residue group (thioether) (top). Oxidation, i.e., the product of the reaction, formed a sulfone residue group on the dGTP (center). The sulfone dGTP was exposed to sodium hydroxide to remove the O-bond residue group from the dGTP. [Figure 41A] This is a capillary electrophoresis ferrogram showing the results of treating oligonucleotides containing a residual N6 carbamate sulfide A group with a base (50 mM NaOH) to remove the residual group and convert the nucleotides containing the residual group to natural adenine. [Figure 41B] This is a capillary electrophoresis ferrogram showing the results of treating oligonucleotides containing a residual N4 carbamate sulfide C group with a base (50 mM NaOH) to remove the residual group and convert the nucleotides containing the residual group back to natural cytosine. [Figure 42]This is a capillary electrophoresis ferrogram showing the results of treating oligonucleotides containing nucleotides with residual N6 carbamate ethyl A groups with a base (50 mM NaOH) to remove the residual groups and convert the nucleotides with residual groups to natural adenine. [Figure 43] The figure shows the results of capillary electrophoresis for i) oligonucleotides containing adenine with an N6-linked residual group and ii) the same oligonucleotides treated with triethylamine (TEA) for 30 minutes. Here, the adenine compounds with an N6-linked residual group are N6 carbamatetopropyl A, N6 carbamate ethyl A, N6 amidopropyl A, and N6 amidoethyl A. [Figure 44] This figure shows the intramolecular cyclization reaction mechanism in which the reaction rate is affected by the ring size of the N-bonded carbamate residual group or protecting group. [Figure 45] This figure shows the rate of deprotection reactions of nucleotides with N-linked residual groups incorporated into oligonucleotides. The nucleotides with residual groups are as follows: N6 carbamate ethyl A (large circle, Et-CO2-A), N6 amidopropyl A (square, Pr-CO-A), N4 carbamate ethyl C (triangle, Et-CO2-C), and N4 carbamate (methyl) ethyl C (small circle, 2MeEt-CO2-C). [Figure 46A] This figure shows the capillary electrophoresis analysis of an uncontrolled oligonucleotide synthesis reaction using dG nucleotides to synthesize a G homopolymer on a 35T starting oligonucleotide (SEQ ID NO: 48). dGTP represents synthesis performed using unmodified nucleotides. O6 sulfone G and O6 sulfide G represent synthesis performed using dGTP nucleotides modified to have a removable protecting group on the base-pairing O6 atom of guanine. The oligonucleotide synthesis reaction was terminated at 30 seconds, 1 minute, 4 minutes, and 8 minutes, and the progress was measured by capillary electrophoresis as shown. [Figure 46B]This figure shows the capillary electrophoresis analysis of an uncontrolled oligonucleotide synthesis reaction using dG nucleotides to synthesize a G homopolymer on a 30C starting oligonucleotide (SEQ ID NO: 50). dGTP represents synthesis performed using unmodified nucleotides. O6 sulfone G and O6 sulfide G represent synthesis performed using dGTP nucleotides modified to have a removable protecting group on the base-pairing O6 atom of guanine. The oligonucleotide synthesis reaction was terminated at 30 seconds, 1 minute, 4 minutes, and 8 minutes, and the progress was measured by capillary electrophoresis as shown. [Figure 47] This figure shows the capillary electrophoresis analysis of an uncontrolled oligonucleotide synthesis reaction using dA nucleotides to synthesize A homopolymer on a 35T starting oligonucleotide (SEQ ID NO: 48). dATP represents synthesis performed using unmodified nucleotides. N6 carbamate ethyl A and N6 carbamate sulfide A represent synthesis performed using dATP nucleotides modified to have a removable protecting group on the base-pairing N6 atom of adenine. The oligonucleotide synthesis reaction was terminated at 30 seconds, 1 minute, 4 minutes, and 8 minutes, and the progress was measured by capillary electrophoresis as shown. [Figure 48] This figure shows two amino acid ester dTTP analogs used for oligonucleotide synthesis and linker cleavage. One is linked by a hydroxypropargyl residual group (linker 1), and the other by a smaller hydroxymethyl residual group (linker 2). The two amino acid ester dTTP analogs (linkers 1 and 2, Figure 48, synthesized by Jena Bioscience) were bonded to a cysteine-reactive crosslinking agent and then to TdT to obtain the final structure shown in Figure 48. The cleavage product with an alcohol residual group after cleavage of the linker ester is also shown. [Figure 49](A-C) These figures show plots of the reaction rates of complex addition to oligonucleotides without residual groups (Figure 49(A and B)) and oligonucleotides with residual hydroxymethyl groups (Figure 49(C)). Figure 49(A): When a native DNA primer is exposed to a dTTP complex containing an ester bond for 1 second, an extension yield of approximately 35% is obtained. Figure 49(B): The oligonucleotide synthesis reaction proceeds to completion, and linker cleavage yields a primer with a residual hydroxymethyl group at the last base. Figure 49(C): When the primer with the residual group is exposed again to a dTTP complex containing an ester bond for 1 second, an extension yield of approximately 35% is obtained. [Figure 50] The results of primer extension using the TdT-dTTP complex with Linker 1 or Linker 2 (measured by gel shift assay on SDS-PAGE) are shown. ssDNA primers were extended for 60 seconds using 1) Linker 1 complex, 2) Linker 2 complex, 3) Linker 2 complex (replicated), or 4) no complex. T / P: TdT / DNA primer complex. P: ssDNA primer. [Figure 51-1] This figure shows primer extension products measured by capillary electrophoresis. Extension was performed using the Linker 2 complex stored overnight at the indicated pH, or simply in buffer (negative control). Extension without insertion shows a peak at approximately 58 nt. A peak indicating unwanted insertion (extension product) is at approximately 59 nt in some samples, indicating the presence of free dNTPs in the incubated complex. [Figure 51-2] See the explanation in Figure 51-1. [Figure 52] This figure (Part A) shows the results of enzymatic synthesis (measured using capillary electrophoresis) of 100-nucleotide and 200-nucleotide dT oligonucleotides (SEQ ID NOs. 51 and 52, respectively) using the linker 2dNTP complex. Part B shows a magnified view of the distribution (measured by capillary electrophoresis) of the enzymatically synthesized 100-nucleotide product (top) and the chemically synthesized 100-nucleotide product (bottom). [Figure 53]This figure shows the results of oligonucleotide elongation and the time course of cleavage. Oligonucleotide elongation was performed using TdT-dATP complex, TdT-dCTP complex, TdT-dGTP complex, and TdT-dTTP complex containing linker 6 (measured by capillary electrophoresis (Panel A)). The time course of cleavage was measured by capillary electrophoresis after incorporating the TdT-dTTP complex containing linker 6 into oligonucleotides and cleaving them with proteinase K for 30 to 240 seconds (Panel B). [Figure 54] This figure shows the structures of linker nucleotides containing glycine amino acid ester (Gly-OMe-U) and ACC amino acid ester (ACC-OMe-U), as well as the product due to the ester instability of both linkers (HOMe-U) (top), and a comparison of the intact product (Gly-OMe-U or ACC-OMe-U) and the hydrolysis product (HOMe-U) after exposure to 45°C for 60 minutes. [Figure 55A] This figure compares the linker cleavage efficiencies of various TdT-nucleotide complexes. [Figure 55B] This figure compares the linker cleavage efficiencies of various TdT-nucleotide complexes. [Figure 55C] This figure compares the linker cleavage efficiency of various TdT-nucleotide complexes. The data shown is from 60 seconds after ProK treatment. [Figure 56-1] A series of electroferrographs showing the cleavage rate by proteinase K (ProK) for exemplary linkers having an aminocyclopropylcarboxyethyl group and one glycine (1XG) or two glycine (2XG) molecules. The cleavage reaction was quenched after 15 seconds (s), 30 seconds, 60 seconds, 4 minutes (m), 8 minutes, or 16 minutes. [Figure 56-2] See the explanation in Figure 56-1. [Figure 57] This figure shows the results of complex attachment to the primer 3.8 seconds after attachment for each TdT-nucleotide complex (L2=ACC, Gly-ACC, or 2XGly-ACC). [Figure 58] This figure shows the ester hydrolysis percentage for compounds 14-18 (a series of ring-expanding linker nucleotides) after exposure to 50°C for 1 minute to 20 hours. [Figure 59-1] This figure shows the results (measured by capillary electrophoresis) of oligonucleotides extended using allyl G complex, ACC complex, AiB complex, AC4C complex, AC5C complex, or AC6C complex after exposure to 50°C for 1 hour, 4 hours, or overnight (showing the ratio of intact material to hydrolysis products). [Figure 59-2] See the explanation in Figure 59-1. [Figure 60A-1] This figure shows the results of exemplary mononucleotide addition reactions to exemplary single-stranded DNA substrates using polymerase complexes A, C, T, or G in the presence or absence of phosphatase (+Phos) or in the absence of phosphatase (-Phos). The resulting synthetic oligonucleotides were analyzed by capillary electrophoresis. The x-axis shows the approximate nucleotide length of the oligonucleotide, and the y-axis shows the relative fluorescence intensity at 517 nm. The reaction was terminated at the indicated point. [Figure 60A-2] Refer to the explanation in Figure 60A-1. [Figure 60B] This figure shows a magnified view of the results at 21 minutes and 41 seconds in Figure 60A, with and without phosphatase. Specific nucleotides are shown in each set of panels. Arrows indicate +2 addition. [Figure 61A-1]The graph shows the results of capillary electrophoresis analysis of mononucleotide addition reactions performed on single-stranded DNA substrates using a T-polymerase complex in the presence of the following exemplary phosphatases: bovine (B. taurus) (Quick CIP, NEB), northern shrimp (P. borealis) (shrimp-derived alkaline phosphatase, NEB), Antarctic bacterium TAB5 (Antarctic phosphatase, NEB), and Escherichia coli (E. coli) (Takara Bio) phosphatase. The synthesis reactions were carried out at room temperature (24°C). A control synthesis reaction was performed without phosphatase. The reaction was terminated at the indicated time. The x-axis shows the relative electrophoresis of oligonucleotides (depending on approximate nucleotide length), and the y-axis shows the relative fluorescence intensity at 517 nm. [Figure 61A-2] Refer to the explanation in Figure 61A-1. [Figure 61B] The graph shows the results of capillary electrophoresis analysis of exemplary mononucleotide addition reactions performed on single-stranded DNA substrates using a T-polymerase complex in the presence of the following exemplary phosphatases: bovine (Quick CIP, NEB), northern shrimp (shrimp-derived alkaline phosphatase, NEB), Antarctic bacterium TAB5 (Antarctic phosphatase, NEB), and Escherichia coli (Takara Bio) phosphatase. The synthesis reactions were carried out at 37°C (with and without phosphatase) and terminated after 30 minutes. Arrows indicate the expected size of the +2 addition. [Figure 62] This graph shows the results of polynucleotide synthesis using exemplary complexes of exemplary 50-nucleotide polynucleotides, performed in the presence or absence of phosphatase. The synthesized polynucleotides are distinguished by size along the x-axis using a SeqStudio gene analyzer. Peaks corresponding to the starting oligonucleotides and the exact 50-nucleotide synthesized products are labeled. [Figure 63A] This is a diagram showing a series of electrophoretic graphs. [Figure 63B] This is a diagram showing a series of electrophoretic graphs. [Figure 63C] This is a diagram showing a series of electrophoretic graphs. [Figure 63D] This figure shows a series of electrophoretic graphs. These show the results of analyzing the products at different time points in an enzymatic polynucleotide elongation reaction using a polymerase-nucleotide complex in a reaction buffer with a low cobalt acetate concentration (0.05 mM CoOAc) or a standard cobalt acetate concentration. [Figure 63E] Figures 63A to 63D show plots of the quantitative analysis of the products and the reaction rates (kobs) calculated for each product. [Figure 64A] This is a diagram showing a series of electrophoretic graphs. [Figure 64B] This is a diagram showing a series of electrophoretic graphs. [Figure 64C] This is a diagram showing a series of electrophoretic graphs. [Figure 64D] This is a diagram showing a series of electrophoretic graphs. [Figure 64E] This is a diagram showing a series of electrophoretic graphs. [Figure 64F] This is a diagram showing a series of electrophoretic graphs. [Figure 64G] This is a diagram showing a series of electrophoretic graphs. [Figure 64H] This is a diagram showing a series of electrophoretic graphs. [Figure 64I] This is a diagram showing a series of electrophoretic graphs. [Figure 64J] This is a diagram showing a series of electrophoretic graphs. [Figure 64K] This is a diagram showing a series of electrophoretic graphs. [Figure 64L] This figure shows a series of electrophoretic graphs. These show the results of analyzing the products at different time points in an enzymatic polynucleotide elongation reaction using a polymerase-nucleotide complex in reaction buffers with cobalt acetate concentrations in a range of 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). [Figure 64M]This figure shows plots of the quantitative analysis of the products in Figures 64A to 64L, along with the calculated reaction rates (kobs). [Figure 65A] This is a diagram showing a series of electrophoretic graphs. [Figure 65B] This is a diagram showing a series of electrophoretic graphs. [Figure 65C] This is a diagram showing a series of electrophoretic graphs. [Figure 65D] This is a diagram showing a series of electrophoretic graphs. [Figure 65E] This is a diagram showing a series of electrophoretic graphs. [Figure 65F] This is a diagram showing a series of electrophoretic graphs. [Figure 65G] This is a diagram showing a series of electrophoretic graphs. [Figure 65H] This is a diagram showing a series of electrophoretic graphs. [Figure 65I] This is a diagram showing a series of electrophoretic graphs. [Figure 65J] This is a diagram showing a series of electrophoretic graphs. [Figure 65K] This is a diagram showing a series of electrophoretic graphs. [Figure 65L] This figure shows a series of electrophoretic graphs. These show the results of analyzing the products at different time points in an enzymatic polynucleotide elongation reaction using a polymerase-nucleotide complex in reaction buffers with a range of zinc acetate 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). [Figure 65M] This figure shows plots of the quantitative analysis of products 65A to 65L, along with the calculated reaction rates (kobs). [Figure 66A] This is a diagram showing a series of electrophoretic graphs. [Figure 66B] This is a diagram showing a series of electrophoretic graphs. [Figure 66C] This is a diagram showing a series of electrophoretic graphs. [Figure 66D] This is a diagram showing a series of electrophoretic graphs. [Figure 66E] This is a diagram showing a series of electrophoretic graphs. [Figure 66F] This is a diagram showing a series of electrophoretic graphs. [Figure 66G] This is a diagram showing a series of electrophoretic graphs. [Figure 66H] This is a diagram showing a series of electrophoretic graphs. [Figure 66I] This is a diagram showing a series of electrophoretic graphs. [Figure 66J] This is a diagram showing a series of electrophoretic graphs. [Figure 66K] This is a diagram showing a series of electrophoretic graphs. [Figure 66L] This figure shows a series of electrophoretic graphs. These show the results of analyzing the products at different time points in an enzymatic polynucleotide elongation reaction using free polymerase and free nucleotides in reaction buffers with cobalt acetate concentrations in a range of 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). [Figure 66M] This plot shows the results of quantitative analysis of the products in Figures 66A to 66L. [Figure 67] This figure shows the percentage of complete polynucleotides at each step of 520-base-length polynucleotide synthesis, as measured by next-generation sequencing. [Figure 68] This figure shows the percentage of complete polynucleotides at each step of 1005-base-length polynucleotide synthesis, as measured by next-generation sequencing. [Figure 69] This graph shows the length and synthesis quality (step yield) of oligonucleotides produced by the process described in Example 35. [Figure 70] This graph shows the length and synthesis quality (step yield) of oligonucleotides longer than 1000 nucleotides produced by the process described in Example 35. [Figure 71]This graph shows the length and synthesis quality of the "all5mer" oligonucleotide sequence produced by the process described in Example 35. [Modes for carrying out the invention]

[0122] Detailed explanation Details of various embodiments of this disclosure are shown in the following description. Other features, purposes, and advantages of this disclosure will become apparent from the description and drawings, as well as the claims.

[0123] definition As described herein, the compounds of this disclosure may include “substituted” moieties. Generally, the term “substituted” means that one or more hydrogens of a specified moiety are replaced by a suitable substituent, whether or not followed by the phrase “may.” Unless otherwise indicated, “substituted” groups may have suitable substituents at each of their substitutable positions, and if multiple positions in any given structure can be replaced by multiple substituents selected from a particular group, such substituents may be the same or different at each position. The substituent combinations envisioned by this disclosure preferably result in the formation of stable or chemically feasible compounds. In this specification, “stable” means that the compound remains substantially unchanged when subjected to conditions that enable production, detection, and, in certain embodiments, recovery, purification, and use for one or more purposes disclosed herein.

[0124] The term "alkyl" refers to a fully saturated hydrocarbon chain, either linear or branched. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.

[0125] The term "haloalkyl" refers to a linear or branched alkyl group that is substituted with one or more halogen atoms.

[0126] As described herein, the compounds of this disclosure may include “substituted” moieties. Generally, the term “substituted” means that one or more hydrogens of a specified moiety are replaced by a suitable substituent, whether or not followed by the phrase “may.” Unless otherwise indicated, “substituted” groups may have suitable substituents at each of their substitutable positions, and if multiple positions in any given structure can be replaced by multiple substituents selected from a particular group, such substituents may be the same or different at each position. The substituent combinations envisioned by this disclosure preferably result in the formation of stable or chemically feasible compounds. In this specification, “stable” means that the compound remains substantially unchanged when subjected to conditions that enable production, detection, and, in certain embodiments, recovery, purification, and use for one or more purposes disclosed herein.

[0127] Suitable monovalent substituents on the substitutable carbon atoms of the "may be substituted" group are, independently, halogens, -(CH2) 0-4 R ○ ,-(CH2) 0-4 Ure ○ -O(CH2) 0-4 R ○ -O-(CH2) 0-4 C(O)OR ○ ,-(CH2) 0-4 CH(OR ○ )2, -(CH2) 0-4 SR ○ ,-(CH2) 0-4 Ph (This is R ○ (May be replaced by) -(CH2) 0-4 O(CH2) 0-1 Ph (This is R ○ (This may be substituted with) -CH=CHPh(This is R ○ (May be replaced by) -(CH2) 0-4 O(CH2) 0-1 - Pyridyl (This is R ○でましますしていています)、-NO2、-CN、-N3、-(CH2) 0-4 N(R ○ )2、-(CH2) 0-4 N(R ○ )C(O)R ○ ,-N(R ○ )C(S)R ○ ,-(CH2) 0-4 N(R ○ )C(O)NR ○ 2、-N(R ○ )C(S)NR ○ 2、-(CH2) 0-4 N(R ○ )C(O)OR ○ ,-N(R ○ )N(R ○ )C(O)R ○ ,-N(R ○ )N(R ○ )C(O)NR ○ 2、-N(R ○ )N(R ○ )C(O)OR ○ ,-(CH2) 0-4 C(O)R ○ 、-C(S)R ○ ,-(CH2) 0-4 C(O)OR ○ ,-(CH2) 0-4 C(O)SR ○ ,-(CH2) 0-4 C(O)OSiR ○ 3、-(CH2) 0-4 OC(O)R ○ 、-OC(O)(CH2) 0-4 SR ○ 、SC(S)SR ○ ,-(CH2) 0-4 SC(O)R ○ ,-(CH2) 0-4 C(O)NR ○ 2、-C(S)NR ○ 2、-C(S)SR ○ ,-SC(S)SR ○ ,-(CH2) 0-4 OC(O)NR ○ 2、-C(O)N(OR ○ )R ○ 、-C(O)C(O)R ○, -C(O)CH2C(O)R ○ , -C(NOR ○ )R ○ , -(CH2) 0-4 SSR ○ , -(CH2) 0-4 S(O)2R ○ , -(CH2) 0-4 S(O)2OR ○ , -(CH2) 0-4 OS(O)2R ○ , -S(O)2NR ○ , -(CH2) 0-4 S(O)R ○ , -N(R ○ )S(O)2NR ○ , -N(R ○ )S(O)2R ○ , -N(OR ○ )R ○ , -C(NH)NR ○ , -P(O)2R ○ , -P(O)R ○ , -OP(O)R ○ , -OP(O)(OR ○ )2, SiR ○ , -(C 1-4 straight-chain or branched-chain alkylene)O-N(R ○ )2, or -(C 1-4 straight-chain or branched-chain alkylene)C(O)O-N(R ○ )2, where each R ○ may be substituted as follows and is independently hydrogen, C 1-6 aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, -CH2-(5-6 member heteroaryl ring), or a 5-6 member saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, regardless of the above, two Rs ○ together with their intervening atom(s) form a 3-12 member saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as follows.

[0128] R ○ (or two independent R ○ Suitable monovalent substituents on the ring formed by the combination of these intervening atoms are, independently, halogens, -(CH2) 0-2 R ● ,-(HaroR ● ), -(CH2) 0-2 OH, -(CH2) 0-2 Ure ● ,-(CH2) 0-2 CH(OR ● )2, -O(HaroR ● ), -CN, -N3, -(CH2) 0-2 C(O)R ● ,-(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR ● ,-(CH2) 0-2 SR ● ,-(CH2) 0-2 SH, -(CH2) 0-2 NH2, -(CH2) 0-2 NHR ● ,-(CH2) 0-2 NR ● 2, -NO2, -SiR ● 3. -OSiR ● 3, -C(O)SR ● ,-(C 1-4 (Linear or branched alkylene) C(O)OR ● , or -SSR ● And here, each R ● It is either unsubstituted, or if preceded by "halo", it is substituted with only one or more halogens, and C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 A 5-6 member saturated ring, partially unsaturated ring, or aryl ring having 0-4 heteroatoms independently selected from Ph, nitrogen, oxygen, or sulfur. ○ Suitable divalent substituents on the saturated carbon atom include =O and =S.

[0129] Suitable divalent substituents on the saturated carbon atom of the "may be substituted" group include =O, =S, =NNR*2, =NNHC(O)R*, =NNHC(O)OR*, =NNHS(O)2R*, =NR*, =NOR*, and -O(C(R*2)). 2-3 O-, or -S(C(R*2)) 2-3 S- is an example, where each independent R* is a hydrogen atom, and C may be substituted as follows. 1-6 Selected from unsubstituted, 5-6 member saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from aliphatic, nitrogen, oxygen, or sulfur. A preferred divalent substituent bonded to a substituted adjacent carbon of the "may be substituted" group is -O(CR*2) 2-3 O- is an example, where each independent R* may be substituted with hydrogen, or C as shown below. 1-6 The rings are selected from unsubstituted, 5-6 member saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms that are aliphatic or independently selected from nitrogen, oxygen, or sulfur.

[0130] Suitable substituents on the aliphatic group of R* include halogens and -R ● ,-(HaroR ● ), -OH, -OR ● ,-O(HaroR ● ), -CN, -C(O)OH, -C(O)OR ● -NH2, -NHR ● , -NR ● 2, or -NO2, where each R ● It is either unsubstituted, or if preceded by "halo", substituted by only one or more halogens, and independently, C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 It is a 5-6 member saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from Ph, nitrogen, oxygen, or sulfur.

[0131] A suitable substituent on the substituted nitrogen of the "optionally substituted" group is -R † , -NR †2, -C(O)R † , -C(O)OR † ,-C(O)C(O)R † -C(O)CH2C(O)R † -S(O)2R † -S(O)2NR † 2, -C(S)NR † 2, -C(NH)NR † 2, or -N(R † )S(O)2R † These are listed, and here, each R † These are, independently, hydrogen, and C which may be substituted as follows. 1-6 An unsubstituted, 5-6 member saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from aliphatic, unsubstituted-OPh, or nitrogen, oxygen, or sulfur, or notwithstanding the above, two independent R † Together with these intervening atoms (multiple atoms are possible), they form an unsubstituted, 3-12 member saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0132] R † Suitable substituents on the aliphatic group are, independently, halogens, -R ● ,-(HaroR ● ), -OH, -OR ● ,-O(HaroR ● ), -CN, -C(O)OH, -C(O)OR ● -NH2, -NHR ● , -NR ● 2, or -NO2, where each R ● It is either unsubstituted, or if preceded by "halo", substituted by only one or more halogens, and independently, C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 It is a 5-6 member saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from Ph, nitrogen, oxygen, or sulfur.

[0133] In this specification, when a variable element is defined by listing chemical groups, the variable element may be a single group from the listed groups or a combination of the listed groups. In this specification, when an embodiment is described for a particular variable element, the embodiment may be used as a standalone embodiment or in combination with other embodiments or parts thereof.

[0134] In another embodiment, the compounds described herein may include one or more isotopic substitutions. For example, hydrogen is 2 H (D or deuterium) or 3 H (or T or tritium) may also be used, and carbon can be, for example, 13 C or 14 It could also be C, and oxygen could be, for example, 18 It could be O, and nitrogen could be, for example, 15 N may also be used, etc. In other embodiments, a specific isotope (for example, 3 H, 13 C, 14 C, 18 O, or 15 N) can account for 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 the element at a particular site of the compound.

[0135] In this specification, the terms “about” or “approximately” mean, as will be apparent to those skilled in the art, that a value or composition is within its acceptable margin of error, which is partly dependent on the method by which the value or composition is measured or determined (i.e., partly dependent on the limits of the measurement system). For example, “about” or “approximately” may mean, according to convention in the art, that a value is within one standard error or more. Alternatively, “about” or “approximately” may mean a range of up to 10% (i.e., ±10%) or more, depending on the limits of the measurement system. For example, about 5 mg can encompass any number between 4.5 mg and 5.5 mg. Also, particularly with respect to biological systems or processes, “about” or “approximately” may mean up to one order of magnitude (10 times) or up to five times a given value. Where a particular value or composition is shown in this disclosure, unless otherwise stated, the meaning of “about” or “approximately” should be understood as being within the acceptable margin of error for that particular value or composition. Also, where a range and / or partial range of a value is shown, that range and / or partial range may include the endpoints of that range.

[0136] In this specification, the terms “nucleic acid,” “polynucleotide,” and “oligonucleotide,” as well as other related terms, are used synonymously and refer to polymers of nucleotides, not limited to any particular length. 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 DNA or RNA produced using nucleotide analogs (e.g., peptide nucleic acids (PNAs) and nucleotide analogs not found in nature), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids contain polymers of nucleotides, where nucleotides contain native or unnatural bases and / or sugars. Nucleic acids contain naturally occurring nucleoside bonds, e.g., phosphodiester bonds. Nucleic acids may lack phosphate groups. Nucleic acids contain unnatural nucleoside bonds. Such bonds include phosphorothioate bonds, phosphorothiolate bonds, or peptide nucleic acid (PNA) bonds. In some embodiments, the nucleic acid comprises one type of polynucleotide or a mixture of two or more different types of polynucleotides.

[0137] In this specification, the terms “operably linked” and “operably linked” or related terms refer to the juxtaposition of components. Juxtaposed components can be covalently linked to one another. For example, two nucleic acid components can be linked to each other enzymatically, where the linkage between the two components includes a phosphodiester bond. A first nucleic acid component and a second nucleic acid component can be linked to each other, where the first nucleic acid component can confer a certain function to the second nucleic acid component. For example, 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. In another example, a transgene (e.g., a nucleic acid encoding a polypeptide, or a nucleic acid sequence of interest) can be linked to a vector, and linkage enables the expression or function of the transgene sequence contained in the vector. In some embodiments, the transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that acts on its expression. In some embodiments, the vector includes at least one host cell regulatory sequence. Such regulatory sequences include promoter sequences, enhancers, transcription and / or translation initiation sequences, transcription and / or translation termination sequences, and polypeptide secretion signal sequences. In some embodiments, host cell regulatory sequences control the level, timing, and / or location of transgene expression.

[0138] The terms “linking,” “joining,” “attachment,” and “addition,” and their variations, encompass any kind of fusion, linking, attachment, or association between any combination of multiple compounds or molecules, such fusions, links, attachments, or associations that are sufficiently stable to withstand use in specific procedures. Such procedures include, but are not limited to, nucleotide linking, nucleotide incorporation, deprotection (unblocking) (e.g., removal of end portions of a chain), washing, removal, flow, detection, imaging, and / or identification. Examples of such linking include, for example, covalent bonds, ionic bonds, hydrogen bonds, dipole bonds, hydrophilic bonds, hydrophobic bonds, or affinity bonds, bonds or associations involving van der Waals forces, and mechanical bonds. In some embodiments, such linking occurs intramolecularly, for example, by linking the ends of single-stranded or double-stranded linear nucleic acid molecules to form a cyclic molecule. In some embodiments, such coupling may occur between combinations of different molecules or between molecules and nonmolecules, and such couplings include, but are not limited to, coupling between nucleic acid molecules and solid surfaces, coupling between proteins and detectable reporter moieties, and coupling between nucleotides and detectable reporter moieties. Some examples of coupling are shown, 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), and Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998).

[0139] In relation to nucleic acids, the terms “extension,” “to elongate,” and “the act of elongation,” and other variations thereof, refer to the incorporation of one or more nucleotides into a nucleic acid molecule (i.e., the bonding of nucleotides). Nucleotide incorporation involves polymerizing one or more nucleotides to the 3'OH end of a nucleic acid chain (e.g., a nucleic acid primer), which results in the elongation of the nucleic acid chain (e.g., a starting oligonucleotide). Nucleotide incorporation can be performed using natural nucleotides and / or nucleotide analogs.

[0140] In this specification, the terms “cleavable linker” or “cleavable portion” refer, respectively, to a divalent or monovalent portion that can be separated into separate parts (e.g., a portion that can be separated, divided, cleaved, hydrolyzed, or has stable bonds within it that can be cleaved). In some embodiments, the cleavable linker is cleavable in response to an external stimulus (e.g., an enzyme, a nucleophilic / basic reagent, a reducing agent, light irradiation, an electrophilic / acidic reagent, an organometallic reagent and a metallic reagent, or an oxidizing reagent) (e.g., specifically cleavable).

[0141] The use of the term "cleavable linker" does not imply that the entire linker must be removed. The cleavage site can be located at a specific point on the linker, where cleavage ensures that a portion of the linker remains bound to the dye and / or substrate. Examples of cleavable linkers include, but are not limited to, electrophilically cleavable, nucleophilically cleavable, photocleavable, cleavable under reducing conditions (e.g., linkers containing disulfides or azides), cleavable under oxidizing conditions, cleavable by safety catch, and cleavable by desorption mechanisms. Using a cleavable linker to bind the dye compound to the substrate allows for the reliable removal of the label after detection, as needed, thus avoiding interference signals in downstream processes.

[0142] In several embodiments, the cutting of a severable linker is performed by bringing it into contact with a cutting agent (a cutting substance) (e.g., a reducing agent). In several embodiments, the cutting agent is...

[0143] In this specification, the terms “polymerase-compatible cleavable moiety” and “polymerase-compatible cleavable linker” refer to a cleavable moiety or linker that does not interfere with the function of a polymerase (e.g., DNA polymerase or modified DNA polymerase that incorporates the nucleotide to which the polymerase-compatible cleavable moiety is bound at the 3' end of a newly formed nucleotide chain). Methods for measuring polymerase function as conceivable herein are described in B. Rosenblum et al. (Nucleic Acids Res. 1997 Nov 15;25(22):4500-4504) and Z. Zhu et al. (Nucleic Acids Res. 1994 Aug 25;22(16):3418-3422). These documents are incorporated herein by reference in their entirety for all purposes. In several embodiments, the polymerase-compatible cleavable moiety does not impair polymerase function compared to its absence. In multiple embodiments, the polymerase-compatible cleavable portion does not adversely affect DNA polymerase recognition. In multiple embodiments, the polymerase-compatible cleavable portion does not adversely affect (e.g., limit) the DNA polymerase read length.

[0144] In this specification, the term “nucleotide” refers to a molecule comprising a nucleoside and one or more phosphate groups. “Nucleoside” refers to a molecule comprising a nucleic acid base (e.g., adenine, thymine, cytosine, guanine, or uracil) and a pentose (e.g., ribose or 2'-deoxyribose). Exemplary nucleotides may be, but are not limited to, nucleoside monophosphate, nucleoside diphosphate, nucleoside triphosphate, nucleoside tetraphosphate, nucleoside pentaphosphate, or nucleoside hexaphosphate, or may contain nucleoside monophosphate, nucleoside diphosphate, nucleoside triphosphate, nucleoside tetraphosphate, nucleoside pentaphosphate, or nucleoside hexaphosphate. The TdT and TdT variants shown herein may, in some embodiments, incorporate any nucleoside polyphosphate (including nucleotide analogs with modifications to nucleic acid bases).

[0145] In this specification, the term "nucleoside polyphosphate" is used to mean "nucleotide," and is sometimes also called "nucleotide polyphosphate." For example, both "nucleotide triphosphate" and "nucleoside triphosphate" refer to nucleotides containing a polyphosphate composed of a nucleic acid base, a sugar, and three linked phosphate groups.

[0146] In this specification, “untermination” or “insertion” occurs when two or more nucleotides are added during a step of cyclic nucleotide elongation. This can occur when an unshielded nucleotide having an uncleaved 5' phosphate is added to an oligonucleotide.

[0147] In this specification, the term “phosphatase” refers to an enzyme capable of removing the 5' phosphate group from a nucleotide (in particular, an unshielded nucleotide as part of an improperly formed complex, or a nucleotide not tethered to a polymerase). Where not a specific phosphatase enzyme is referred to herein, “phosphatase” is intended to encompass all phosphatase enzymes, engineered enzymes having phosphatase activity, or functional fragments thereof capable of removing one or more phosphate groups from a nucleotide. Furthermore, “phosphatase” can refer to any biomolecule (e.g., polypeptide or ribozyme) capable of removing one or more phosphate groups from a nucleotide, and such biomolecules include engineered enzymes having phosphatase activity or functional fragments thereof.

[0148] In this specification, the terms “protected nucleotide” or “shielded nucleotide” refer to a nucleotide whose 5' phosphate removal ability by a phosphatase is sterically inhibited by a tethered polymerase (or other entity or component, such as a protecting group). In some embodiments, such nucleotides are likely to inhibit subsequent nucleotide addition after being attached to an oligonucleotide until the tethered polymerase is removed.

[0149] In this specification, the terms “unprotected nucleotide” or “unshielded nucleotide” refer to a nucleotide whose 5' phosphate removal ability by a phosphatase is not sterically hindered by a tethered polymerase (or other entity or component, such as a protecting group). In some embodiments, an unshielded nucleotide may be tethered to a polymerase (e.g., to a misfolded polymerase) or tethered to a misposition. An unshielded nucleotide may not be tethered to a polymerase (or may be free). If an unshielded nucleotide is not exposed to a phosphatase, it is more likely to be misattached to a polynucleotide as an insert after a shielded nucleotide has been properly attached.

[0150] Overview - Synthesis of long polynucleotides One of the major challenges in the field of polynucleotide synthesis is the de novo synthesis of long polynucleotide sequences. Because nucleotides are added one at a time during the synthesis process, it can be difficult to synthesize very long sequences accurately and efficiently without errors. Even small step errors during synthesis can quickly accumulate and hinder the synthesis of long sequences.

[0151] This specification describes reagents, systems, and methods for the stepwise, template-independent de novo synthesis of high-quality long polynucleotides, for example, using enzymatic polynucleotide synthesis.

[0152] In particular, this specification describes improved reagents, systems, and methods for stepwise polynucleotide synthesis. Such improved reagents, systems, and methods reduce common errors during synthetic reactions (e.g., insertion of unwanted nucleotides (e.g., due to the addition of imperfectly protected nucleotides to an elongating polynucleotide chain, or due to premature removal of protecting groups during the elongation reaction) or deletion of unwanted nucleotides (e.g., due to incomplete reactions (e.g., failure to add a nucleotide during the elongation reaction, or failure to remove a protecting group during the deprotection reaction of a nucleotide)).

[0153] In some embodiments, modified nucleotides that inhibit secondary structure formation in enzymatic polynucleotide synthesis are provided herein. Such secondary structure formation may inhibit polynucleotide elongation by certain polymerases, such as TdT. In some embodiments, conditions for reagents and cofactors optimized to improve the activity of enzymatic polynucleotide synthesis are provided herein. Such improved reagents and methods suppress deletions resulting from incomplete elongation, thereby improving the step yield of long polynucleotide synthesis.

[0154] In some embodiments, reagents for suppressing unwanted insertions are provided herein (for example, phosphatase treatment of nucleotide-linker complexes, which suppresses the activity of unprotected nucleotides during the extension step).

[0155] In some embodiments, improved linkers between a protecting group (e.g., TdT) and a nucleotide are provided herein. Such linkers are stable during the extension step (to suppress unwanted insertions) but are rapidly and completely cleaved during the protecting group removal step (to suppress unwanted deletions).

[0156] Furthermore, in several embodiments, this specification provides improved systems for oligonucleotide synthesis (for example, systems that improve synthesis rate and reagent delivery by immersing the synthesis surface in appropriate pre-prepared extension buffer, protective group removal buffer, and wash buffer). Such systems can also function to improve overall step yield and reaction cycle rate.

[0157] In some embodiments, this specification provides a template-independent de novo synthesis method for polynucleotides of at least 500 bp in length, with an error rate of less than 1% compared to a predetermined sequence.

[0158] In some embodiments, the de novo-synthesized long polynucleotide is at least 600 nucleotides long, at least 700 nucleotides long, at least 800 nucleotides long, at least 900 nucleotides long, or at least 1000 nucleotides long. In some embodiments, the de novo-synthesized long polynucleotide is 500 to 1000 nucleotides long.

[0159] In some embodiments, the error rates observed for each bond during template-independent de novo synthesis of long polynucleotides are 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.

[0160] In some embodiments, the step yields observed for template-independent de novo synthesis of long polynucleotides are 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 step yields observed for template-independent de novo synthesis of long polynucleotides are between 99% and 99.9%.

[0161] In some embodiments, each nucleotide conjugation step in the template-independent de novo synthesis of long polynucleotides 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 conjugation step in the template-independent de novo synthesis of long polynucleotides 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. In some embodiments, nucleotide conjugation to obtain the de novo-synthesized polynucleotide is performed at a rate of 5 nucleotides per hour to 25 nucleotides per hour.

[0162] In some embodiments, template-independent de novo synthesis of polynucleotides produces at least 10 copies, at least 20 copies, at least 50 copies, at least 100 copies, at least 500 copies, at least 1000 copies, at least 2000 copies, or at least 5000 copies of each sequence.

[0163] For each oligonucleotide synthesized on the substrate, the total error rate (multiple) for each type of error (e.g., deletion, insertion, or substitution) is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or higher for the oligonucleotides synthesized on the substrate; or, on average for the substrate, at a maximum of 1:100, 1:500, 1:1000, 1:10000, 1:20000. It can be 1:30000, 1:40000, 1:50000, 1:60000, 1:70000, 1:80000, 1:90000, 1:1000000, or less, or at most approximately 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. For each oligonucleotide synthesized on the substrate, the total error rate (multiple) for each type of error (e.g., deletion, insertion, or substitution) can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or higher, or on average for the substrate, 1:100 to 1:10000, 1:500 to 1:30000. As will be apparent to those skilled in the art, the total error rate (or number of errors) for each type of error (e.g., deletion, insertion, or substitution) for each oligonucleotide synthesized on a substrate may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or higher for the oligonucleotides synthesized on the substrate, or at any of the above values ​​on average, for example, between 1:500 and 1:10000.For each oligonucleotide synthesized on the substrate, the total error rate(s) for each type of error (e.g., deletion, insertion, or substitution) may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or higher, or on average for the substrate, within a range determined by any endpoint among the above values.

[0164] The required predetermined sequence can be supplied by any means, typically by the user (for example, by a user inputting data using a computerized system). In various embodiments, the synthesized nucleic acid is compared with such predetermined sequence. In some cases, this is done by sequencing at least a portion of the synthesized nucleic acid, for example, using next-generation sequencing.

[0165] Polynucleotides can be released from a substrate by various suitable methods, which are described in more detail elsewhere in this specification, and by various suitable methods known in the art (e.g., enzymatic cleavage known in the art). Examples of such enzymatic cleavage include the use of restriction enzymes such as MIyI, and the use of other enzymes or combinations of enzymes capable of cleaving single-stranded or double-stranded DNA (e.g., uracil DNA glycosylase (UDG) and DNA endonuclease IV), without limitation. Other cleavage methods known in the art may also be advantageously used in this disclosure. Such cleavage methods include, but are not limited to, chemical cleavage (destabilization by basic conditions) or optical cleavage (destabilization by light irradiation) of DNA molecules from the surface. Alternatively, constructs for gene synthesis can be generated by replicating oligonucleotides while they are still immobilized on the substrate using PCR or other amplification reactions. Methods for releasing polynucleotides are described in PCT Patent Publication WO2007137242 and U.S. Patent No. 5,750,672, which are incorporated herein by reference in their entirety.

[0166] Enzymatic polynucleotide synthesis In some embodiments, a method for synthesizing polynucleotides of desired length and sequence is provided herein, according to embodiments described herein. In some embodiments, the process is carried out by immersing a reaction surface having bound synthetic starting materials (which may include pre-added nucleotides) in a containment solution containing the necessary reagents. The method is suitable for multiple polynucleotide synthesis and therefore can use a plurality of components having terminals with a reaction surface and bound synthetic starting materials, and such a plurality of components can be simultaneously immersed in a plurality of contained liquid reagents (e.g., present in wells or droplets on the surface) aligned with the reaction surface. In some embodiments, the contained liquid reagents include a polymer extension solution, which includes a particular type of nucleotide and a polymerase capable of adding the nucleotide to the synthetic starting materials.

[0167] In some embodiments, the synthesis of polynucleotides involves a stepwise addition of protected nucleotides to oligonucleotides bound to a reaction surface on a component by repeatedly performing the following steps: adding a nucleotide containing a protecting group (i.e., a protected nucleotide) to a synthetic starting material or an elongated polynucleotide containing a pre-added nucleotide; attaching the nucleotide to the end of the synthetic starting material or the elongated polynucleotide by catalytic action of a polymerase; and removing the protecting group from the nucleotide so that subsequent nucleotides can be added to the elongated polynucleotide. These steps can be repeated until a desired polynucleotide sequence and length are synthesized.

[0168] Protecting groups attached to nucleotides (e.g., polymerases or reversible terminators) are groups that, once a nucleotide is added to a synthetic starting material or an elongated polynucleotide, can prevent the addition of another nucleotide. During the elongation cycle, the required protected nucleotides are added, and after removing excess nucleotides, the elongated polynucleotide is immersed in a nucleotide deprotection solution that can remove the protecting groups from the nucleotides.

[0169] In some embodiments, the protecting group is a polymerase that catalyzes the addition of a nucleotide to a surface-bound polynucleotide, where the polymerase is linked to the nucleotide (i.e., forms a nucleotide-polymerase complex). In this embodiment, the polymerase can sterically interfere with the addition of subsequent nucleotides after the protected nucleotide has been added to the polynucleotide. The protecting group can then be removed using a monomer deprotection solution (e.g., a linker cleavage solution) that removes the polymerase from the nucleotide.

[0170] In some embodiments, the protecting group is a reversible terminator bound to the nucleotide. The protecting group can then be removed using a monomer deprotection solution (e.g., a linker cleavage solution) that removes the reversible terminator from the nucleotide. In some embodiments, both a reversible terminator bound to the nucleotide and a polymerase may be used.

[0171] Both the nucleotide addition step and the protecting group removal step can be stopped by immersing the extended polynucleotide in a suitable reaction stop solution such as EDTA. Furthermore, a washing step may be used between steps by immersing the extended polynucleotide in a washing buffer.

[0172] Polynucleotide synthesis by complex In some embodiments, the present disclosure uses TdT with a free nucleotide having a 3' modification to enable a single extension. Also in some embodiments, the present disclosure uses TdT having a tethered nucleotide (which the inventors refer to as a polymerase-nucleotide complex). Binding of the dNTP can be carried out via a tethering chain to the nucleic acid base. In some embodiments, the nucleotide has an O-alkyl group which may be substituted. Further examples of tethered creotides are described, for example, in PCT Publication WO2017 / 223517, “Nucleic Acid Synthesis and Sequencing Using Tethered Nucleoside Triphosphates,” the entire document of which is incorporated by reference.

[0173] This specification describes a method for the de novo synthesis of nucleic acids using a complex comprising a polymerase and a nucleoside triphosphate. In some embodiments of the method, the complex comprises polymerase terminal deoxynucleotidyltransferase (TdT). In other embodiments, the method may use a complex comprising a different template-independent polymerase.

[0174] Figure 1A shows a typical process for stepwise synthesis of a given sequence using template-independent polymerase. A nucleic acid acting as the initial substrate for extension (i.e., the "starting molecule") is incubated with a first polymerase-nucleotide complex. Once the nucleic acid is extended by the nucleotides linked by the complex, no further extension occurs as the complex executes a termination mechanism. In the second step of the process, the linker is cleaved, releasing the polymerase and reversing the termination mechanism, thereby enabling further extension. The extension product is then exposed to the second complex, and these two steps are repeated to extend the nucleic acid by the desired sequence. Figure 1B shows a synthesis procedure using a complex containing TdT and a photocleavable linker. As mentioned above, other methods are also available for linker binding and cleavage.

[0175] For RNA synthesis applications, tethered ribonucleoside triphosphates can be used. In such embodiments, RNA-specific nucleotidyltransferases (e.g., particularly E. coli poly(A) polymerase (IUBMB EC2.7.7.19) or poly(U) polymerase) can be used. RNA nucleotidyltransferases can be modified (e.g., single point mutations) that affect substrate specificity to particular rNTPs (Lunde et al., Nucleic acids research 40.19(2012):9815-9824). In some embodiments, using a very short tethering chain between the RNA nucleotidyltransferase and the ribonucleoside triphosphate allows for a high effective concentration of nucleoside triphosphate, thereby promoting the incorporation of rNTPs that are not naturally occurring substrates of the nucleotidyltransferase.

[0176] When a complex containing polymerase and nucleoside triphosphate is incubated with nucleic acid, the complex preferentially elongates the nucleic acid by utilizing its tethered nucleotide (rather than using nucleotides from other complex molecules). As described above, the polymerase then remains bound to the nucleic acid via its tethering chain to the attached nucleotide until it is exposed to some stimulus that causes the bond to the attached nucleotide to be cleaved. In this situation, further elongation by the polymerase-nucleotide complex is prevented by "shielding" if 1) the bound polymerase molecule interferes with other complexes, preventing it from approaching the 3'OH of the elongating DNA molecule, and 2) other nucleoside triphosphates in the system interfere with the catalytic site of the polymerase that remains bound to the 3' end of the elongating nucleic acid. (The degree of shielding can be expressed as the degree to which both of these interactions are interfered with). To enable further extension, the linker that ligates the incorporated nucleotide to the polymerase can be cleaved, releasing the polymerase from the nucleic acid and thereby re-exposing its 3'OH group for further extension.

[0177] The method for nucleic acid synthesis described herein utilizes this shielding effect that leads to termination. This method includes an extension step, in which the nucleic acid is preferentially exposed to the complex in the absence of free (i.e., untethered) nucleoside triphosphates, because the shielding termination mechanism may not prevent the incorporation of nucleoside triphosphates into the nucleic acid.

[0178] In some embodiments, the termination of further elongation can be "complete." This means that no further elongation can occur during the reaction after the nucleic acid molecule has been elongated by the complex. In other embodiments, the termination of further elongation may be "incomplete." This means that further elongation can occur during the reaction, but only at a significantly slower rate than the initial elongation (e.g., 100 times slower, or 1000 times slower, or 10,000 times slower, or even slower). If the reaction is stopped after a suitable time, a complex resulting in an incomplete termination may be used to elongate the nucleic acid mainly by a single nucleotide (e.g., in methods for nucleic acid synthesis and sequencing). In some embodiments, the reagent containing the complex may further contain polymerase without the tethered nucleoside triphosphate, but such polymerase should not have a significant effect on the reaction because there is no free dNTP in the mixture.

[0179] Reagents based on complexes that utilize shielding effects to terminate preferentially contain only polymerase-nucleotide complexes in which all polymerases are folded in an active conformation. In some cases, if the polymerase portion of the complex is not folded, its tethered nucleoside triphosphate may be more easily accessible to the polymerase portion of other complex molecules. In such cases, the unshielded nucleotide becomes more easily incorporated by other complex molecules and escapes the termination mechanism.

[0180] Polymerase-nucleotide complexes that utilize shielding effects to terminate the reaction should preferentially be conjugated with only a single nucleoside triphosphate moiety. Polymerase-nucleotide complexes conjugated with multiple nucleoside triphosphates that can access the catalytic site may, in some cases, incorporate multiple nucleoside triphosphates into the same nucleic acid. Therefore, additional tethered nucleoside triphosphates may lead to the undesirable incorporation of further nucleotides into the nucleic acid during the reaction. Furthermore, since only one tethered nucleoside triphosphate can occupy the (embedded) catalytic site of that polymerase at a time, the presence of other tethered nucleoside triphosphates may increase the likelihood of access to the polymerase moiety of other complex molecules, as described below.

[0181] Polymerase-nucleotide complexes that utilize a shielding effect to terminate, enabling rapid integration of nucleotides into nucleic acids, preferentially include the shortest possible linker, allowing frequent access to the catalytic site of the tethered polymerase molecule in an effective conformation. Such complexes also allow the linker's binding site to the polymerase to be preferentially as close as possible to the catalytic site, thereby enabling the use of shorter linkers. The linker length determines the maximum distance from the binding site that the tethered nucleoside triphosphate or tethered nucleic acid can reach. Shorter distances may reduce the likelihood of the tethered portion accessing other polymerase-nucleotide molecules, as will be discussed later. In some embodiments, the linkers are approximately 24 Å and 28 Å long. Shorter linkers, e.g., 8–15 Å long, may increase shielding, while longer linkers, e.g., longer than 50 Å, 70 Å, or 100 Å, may reduce shielding. A combination of multiple factors can influence the shielding effect. Such factors include, but are not limited to, the structure of the polymerase, the length of the linker, the linker's binding site 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.

[0182] One possible contributing factor to shielding is steric effects. Such steric effects block the 3'OH group of the nucleic acid extended by the complex, preventing it from reaching the catalytic site of the polymerase moiety of another complex. Steric effects can also interfere with the tethered nucleoside triphosphate, preventing it from reaching the catalytic site of another polymerase-nucleotide complex molecule through collisions between the complexes that would occur during approach. If such steric effects completely block the effective interaction between the tethered nucleoside triphosphate (or extended nucleic acid) of one complex molecule and the other complex molecule, then such steric effects can lead to complete termination. On the other hand, if such intermolecular interactions are simply interfered with, then such steric effects can lead to incomplete termination.

[0183] Another factor contributing to shielding is the binding affinity of the tethered nucleoside triphosphate to the catalytic site of the polymerase. The tethered nucleoside triphosphate of the complex has a high effective concentration at the catalytic site of the polymerase it tethers, allowing it to remain bound to that site for a long time. When the nucleoside triphosphate is bound to the catalytic site of the polymerase molecule it tethers, it is not available for incorporation by other polymerase molecules. Therefore, tethering reduces the effective concentration of nucleoside triphosphate available for intermolecular incorporation (i.e., incorporation of nucleotides catalyzed by polymerase molecules that are not tethered). This shielding effect can enhance termination by using the nucleoside triphosphate moiety of one complex molecule to reduce the rate at which the polymerase moiety of another complex molecule extends the nucleic acid.

[0184] Another factor contributing to shielding is the binding affinity of the 3' region of the nucleic acid molecule to the catalytic site of the polymerase molecule. After extension by the complex, the nucleic acid is tethered to the complex via its 3' terminal nucleotide, and because it has a high effective concentration at the catalytic site of the polymerase it is tethered to, the nucleic acid can remain bound to that site for a long time. When the nucleic acid is bound to the catalytic site of the polymerase molecule it is tethered to, it is not available for extension by other complex molecules. This effect can enhance termination by reducing the rate at which the nucleic acid extended by the first complex is further extended by other complex molecules.

[0185] In some embodiments, the polymerase-nucleotide complex includes further parts that sterically prevent the tethered nucleoside triphosphate (or the tethered nucleic acid after extension) from approaching the catalytic site of another complex molecule. Such parts include polypeptide or protein domains that can be inserted into the polymerase loop, and further, bulky molecules such as polymers that can site-specifically bind to, for example, an inserted non-natural amino acid or a specific polypeptide tag.

[0186] In some embodiments, the linker is bonded to the 5-position of the pyrimidine or the 7-position of 7-deazaprine. In other embodiments, the linker may be bonded to the extracyclic amine of the nucleic acid base by N-alkylating the extracyclic amine of cytosine at the nitrobenzyl moiety, for example, as described below. In other embodiments, the linker may be bonded to any other atom in the nucleic acid base, sugar, or oc-phosphate, as will be obvious to those skilled in the art.

[0187] Certain polymerases exhibit high tolerance to modifications at specific parts of nucleotides. For example, modifications at position 5 of pyrimidines and position 7 of purines are well tolerated by several polymerases (He and Seela, Nucleic Acids Research 30.24(2002):5485-5496, or Hottin et al., Chemistry. 2017 Feb 10;23(9):2109-2118). In some embodiments, linkers are bound to these positions.

[0188] Preparation of polymerase-nucleotide complexes In some embodiments, the polymerase-nucleotide complex is prepared by first synthesizing an intermediate compound containing a linker and a nucleoside triphosphate (hereinafter referred to as "linker-nucleotide"), and then binding the intermediate compound to a polymerase.

[0189] In some cases, nucleosides with substitutions relative to natural nucleosides (e.g., pyrimidines with a 5-hydroxymethyl substituent or a 5-propargylamino substituent, or 7-deazaprines with a 7-hydroxymethyl substituent or a 7-propargylamino substituent) can serve as useful starting materials for the preparation of linker nucleotides. A set of exemplary nucleosides having 5-hydroxymethyl and 7-hydroxymethyl substituents that may be useful for the preparation of linker nucleotides are shown below. TIFF2026522421000024.tif26165

[0190] A series of exemplary nucleosides having 5-deaza-7-propargylamino substituents and 7-deaza-7-propargylamino substituents that may be useful for the preparation of linker nucleotides are shown below. TIFF2026522421000025.tif29165

[0191] These nucleosides are also commercially available as deoxyribonucleoside triphosphates.

[0192] In some embodiments, sulfhydryl-specific binding groups can be used to specifically conjugate nucleotides to cysteine ​​residues of polymerases. Possible sulfhydryl-specific binding groups include, but are not limited to, ortho-pyridyl disulfide (OPSS), maleimide functional groups, 3-arylpropioronitrile functional groups, arenamide functional groups, haloacetyl functional groups (e.g., iodoacetyl or bromoacetyl), alkyl halides, or perfluoroaryl groups that react well with sulfhydryls surrounded by specific amino acid sequences (Zhang, Chi, et al. Nature Chemistry 8, (2015): 120-128). Other binding groups for the specific conjugation of cysteine ​​residues are obvious to those skilled in the art or are described in relevant literature and texts (e.g., Kim, Younggyu, et al. Bioconjugate Chemistry 19.3 (2008): 786-791).

[0193] In other embodiments, the linker can be bound to the lysine residue via an amine-reactive functional group (e.g., NHS esters, sulfo-NHS esters, tetrafluorophenyl or pentafluorophenyl esters, isothiocyanates, sulfonyl chlorides, etc.). In other embodiments, the linker may be bound to the polymerase via binding to a genetically modified non-natural amino acid. Such non-natural amino acids include, for example, p-propargyloxyphenylalanine or p-azidophenylalanine, which can undergo azido-alkyne Huisgen ring addition. However, many non-natural amino acids suitable for site-directed addition exist and are described in the literature (e.g., Lang and Chin., Chemical Reviews 114.9 (2014):4764-4806).

[0194] In other embodiments, the linker may be specifically bound to the N-terminus of the polymerase. In some embodiments, the polymerase is mutated to have an N-terminal serine or threonine residue, which can be specifically oxidized to produce an N-terminal aldehyde, which can then be bound to, for example, a hydrazide. In other embodiments, the polymerase is mutated to have an N-terminal cysteine ​​residue, to which an aldehyde can be specifically conjugated to form a thiazolidinedion. In other embodiments, a peptide linker can be conjugated to the N-terminal cysteine ​​residue by native chemical ligation.

[0195] In other embodiments, peptide tag sequences can be inserted into polymerases that can be specifically conjugated with synthetic groups by enzymes. Enzymatic conjugation can be performed, for example, using biotin ligase, transglutaminase, lipoic acid ligase, bacterial saltase, and phosphopantetheinyltransferase, as described in the literature (e.g., Stephanopoulos & Francis Nat. Chem. Biol. 7, (2011) 876-884, references 74-78).

[0196] In other embodiments, a linker is attached to a label domain fused to a polymerase. For example, a linker having the corresponding reactive moiety can be used to covalently confer SNAP tags, CLIP tags, HaloTag, and acyl carrier protein domains (see, for example, Stephanopoulos & Francis Nat. Chem, Biol. 7, (2011) 876-884, references 79-82).

[0197] In other embodiments, as described by Carrico et al. (Nat. Chem. Biol. 3, (2007) 321-322), a linker is attached to an aldehyde specifically generated within the polymerase. For example, an amino acid sequence recognized by the enzyme formylglycine-producing enzyme (FGE) may be inserted into the polymerase, and then it may be exposed to FGE. FGE specifically converts the cysteine ​​residue in the recognition sequence to formylglycine (i.e., generates an aldehyde). Then, for example, a hydrazide or aminooxy moiety of a linker can be specifically attached to this aldehyde.

[0198] In some embodiments, the linker may be bonded to the polymerase by a non-covalent bond. Such a non-covalent bond is the bond of one portion of the linker to the portion fused to the polymerase. Examples of such bonding methods include fusing the polymerase to streptavidin, which can bind to the biotin portion of the linker, or fusing the polymerase to anti-digoxigenin, which can bind to the digoxigenin portion of the linker. In some embodiments, the linker's attachment to the polymerase can be brought about by site-directed conjugation, which can be easily reversed (e.g., an ortho-pyridyl disulfide (OPSS) group, which forms a disulfide bond with cysteine, which can be cleaved using a reducing agent (e.g., using TCEP)). Other binding chemical groups, on the other hand, form permanent bonds.

[0199] In any embodiment, as will be apparent to those skilled in the art, the polymerase may be mutated to specifically and reliably bind anchoring nucleotides to particular positions on the polymerase. For example, with sulfhydryl-specific binding chemical groups such as maleimide or ortho-pyridyl disulfide, accessible cysteine ​​residues in the wild-type polymerase can be mutated to non-cysteine ​​residues to prevent denaturation at such positions. In this "reactive cysteine-free" background, cysteine ​​residues can be introduced by mutation to the desired binding sites. Such mutations are selective and do not interfere with polymerase activity.

[0200] Other strategies for site-specifically attaching synthetic groups to proteins are obvious to those skilled in the art and are outlined in the literature (e.g., Stephanopoulos & Francis Nat. Chem. Biol. 7, (2011) 876-884).

[0201] In some embodiments, the polymerase-nucleotide complex is prepared by first synthesizing an intermediate compound containing a linker and a nucleotide (hereinafter referred to as "linker-nucleotide"), and then binding this intermediate compound to a polymerase.

[0202] As will be apparent to those skilled in the art, the complexes and nucleotides disclosed herein can be prepared in a manner similar to the reaction schemes shown below. A rough synthetic approach using such reaction schemes can be shown for specific nucleotides. Similar synthetic approaches can be applied to relevant nucleotide analogs.

[0203] There are several known reactions and functional groups suitable for the formation of nucleotide-polymerase complexes having cleavable linkers conforming to those described herein. For example, linking of the complex components can be achieved by disulfide formation (formation of easily cleavable linkages), amide formation, ester formation, protein-ligand bonding (e.g., biotin-streptavidin bonding), alkylation (e.g., using substituted iodoacetamide reagents), or by forming adducts using aldehydes and amines or hydrazines.

[0204] In some embodiments, each component of the complex contains a site (i.e., a binding group) suitable for binding to facilitate complex synthesis. Examples of such binding groups include, but are not limited to, hydroxyls, esters, amines, carbonates, acetals, aldehydes, aldehyde hydrates, alkenyls, acrylates, methacrylates, acrylamides, activated sulfones, hydrazides, thiols, alkan acids, acid halides, isocyanates, isothiocyanates, maleimides, vinyl sulfones, dithiopyridines, vinylpyridines, iodoacetamides, epoxides, glyoxal, diones, mesylates, tosylates, and toresylates.

[0205] Further examples of binding groups include -NH2, -COOH, -COOCH3, -N-hydroxysuccinimide, and -maleimide. In some embodiments, the biocompatible reactive group may be protected (e.g., by a protecting group). Further examples of biocompatible reactive groups and the resulting biocompatible reactive linkers can be found, for example, in PCT Publication WO2021 / 226327 (incorporated by reference in whole).

[0206] Exemplary reaction schemes for preparing linker-nucleotide complexes having a linker containing an amino acid ester (e.g., the linker described herein and its modifications thereof) are described herein. In some embodiments, the required nucleotides are commercially available, their hydroxyl groups are protected with TBS, and then the L1-OH group is bonded to an extracyclic oxygen or amine on the nucleic acid base. Alternatively, pre-modified nucleotides having an L1-OH group bonded to the nucleotide (e.g., an L1-OH group bonded to C5 of a pyrimidine, or an L1-OH group bonded to C7 of a 7-deazapurine) can be obtained. Then, an Fmoc-protected amino acid ester is bonded to the hydroxyl group of the L1-OH, followed by deprotection of the hydroxyl group and triphosphorylation of the nucleoside.

[0207] To complete the linker, Fmoc is removed from the amino acid ester amine group, and the product is attached to the remainder of the linker containing L3, which can be bound to polymerase.

[0208] In some embodiments, the linker is attached to an atom in the nucleotide that does not participate in base pairing. In some embodiments, the linker is attached to an atom in the nucleic acid base of the nucleotide that is involved in base pairing. In some embodiments, the linker is located at least on an atom that links the polymerase to any atom of a monocyclic or polycyclic system (e.g., pyrimidine or purine or 7-deazapurine or 8-aza-7-deazapurine) bonded at the Γ position of a sugar.

[0209] Certain polymerases exhibit high tolerance to modifications of specific parts of nucleotides. For example, modifications at position 5 of pyrimidines and position 7 of purines are well tolerated by several polymerases (He and Seela, Nucleic Acids Research 30.24(2002):5485-5496, or Hottin et al., Chemistry. 2017 Feb 10;23(9):2109-2118). In some embodiments, the linker is attached to position 5 of pyrimidines or position 7 of 7-deazapurines. In other embodiments, the linker may be attached to the extracyclic amine of the nucleic acid base by N-alkylation of the extracyclic amine of cytosine at the nitrobenzyl moiety, for example, as described below.

[0210] In some embodiments, the linker is attached to the sugar or α-phosphate of the nucleotide. In some embodiments, the linker is attached to the terminal phosphate of the nucleotide. In all embodiments, it is desirable that the linker used be long enough so that the nucleotide can access the active site of the polymerase (to which the nucleotide is tethered). As shown in more detail below, the polymerase of the complex can catalyze the reaction to add the nucleotide to which it is tethered to the 3' end of the nucleic acid.

[0211] The bonding of nucleotides or other base-pairing moieties to a linker can be achieved by any means known in the art of chemical bonding. Nucleotide bases can be obtained or modified to include the L1 portion of the linker. The remaining portion of the linker can be bonded to L1 using the methods illustrated herein. Suitable methods for bonding the linker based on the reactivity of such bases are apparent to or can be determined by those skilled in the art.

[0212] In some embodiments, nucleotides having a base modification to add a free amine group may be used for binding to the linker described herein. For example, a primary amine can be bonded to a base so that it can be reacted with a heterobifunctional polyethylene glycol (PEG) linker to create a nucleotide containing a variable-length PEG linker. Examples of such amine-containing nucleotides include 5-propargylamino-dNTPs, 5-propargylamino-NTPs, aminoallyl-dNTPs, and aminoallyl-NTPs.

[0213] In some embodiments, sulfhydryl-specific binding groups can be used to specifically conjugate nucleotides to cysteine ​​residues of polymerases. Possible sulfhydryl-specific binding groups include, but are not limited to, ortho-pyridyl disulfide (OPSS), maleimide functional groups, 3-arylpropioronitrile functional groups, arenamide functional groups, haloacetyl functional groups (e.g., iodoacetyl or bromoacetyl), alkyl halides, or perfluoroaryl groups that react well with sulfhydryls surrounded by specific amino acid sequences (Zhang, Chi, et al. Nature Chemistry 8, (2015): 120-128). Other binding groups for the specific conjugation of cysteine ​​residues are obvious to those skilled in the art or are described in relevant literature and texts (e.g., Kim, Younggyu, et al. Bioconjugate Chemistry 19.3 (2008): 786-791).

[0214] In other embodiments, the linker can be bound to the lysine residue via an amine-reactive functional group (e.g., NHS esters, sulfo-NHS esters, tetrafluorophenyl or pentafluorophenyl esters, isothiocyanates, sulfonyl chlorides, etc.). In other embodiments, the linker may be bound to the polymerase via binding to a genetically modified non-natural amino acid. Such non-natural amino acids include, for example, p-propargyloxyphenylalanine or p-azidophenylalanine, which can undergo azido-alkyne Huisgen ring addition. However, many non-natural amino acids suitable for site-directed addition exist and are described in the literature (e.g., Lang and Chin., Chemical Reviews 114.9 (2014):4764-4806).

[0215] In other embodiments, the linker may be specifically bound to the N-terminus of the polymerase. In some embodiments, the polymerase is mutated to have an N-terminal serine or threonine residue, which can be specifically oxidized to produce an N-terminal aldehyde, which can then be bound to, for example, a hydrazide. In other embodiments, the polymerase is mutated to have an N-terminal cysteine ​​residue, to which an aldehyde can be specifically conjugated to form a thiazolidinedion. In other embodiments, a peptide linker can be conjugated to the N-terminal cysteine ​​residue by native chemical ligation.

[0216] In other embodiments, peptide tag sequences can be inserted into polymerases that can be specifically conjugated with synthetic groups by enzymes. Enzymatic conjugation can be performed, for example, using biotin ligase, transglutaminase, lipoic acid ligase, bacterial saltase, and phosphopantetheinyltransferase, as described in the literature (e.g., Stephanopoulos & Francis Nat. Chem. Biol. 7, (2011) 876-884, references 74-78).

[0217] In other embodiments, a linker is attached to a label domain fused to a polymerase. For example, a linker having the corresponding reactive moiety can be used to covalently confer SNAP tags, CLIP tags, HaloTag, and acyl carrier protein domains (see, for example, Stephanopoulos & Francis Nat. Chem, Biol. 7, (2011) 876-884, references 79-82).

[0218] In other embodiments, as described by Carrico et al. (Nat. Chem. Biol. 3, (2007) 321-322), a linker is attached to an aldehyde specifically generated within the polymerase. For example, an amino acid sequence recognized by the enzyme formylglycine-producing enzyme (FGE) may be inserted into the polymerase, and then it may be exposed to FGE. FGE specifically converts the cysteine ​​residue in the recognition sequence to formylglycine (i.e., generates an aldehyde). Then, for example, a hydrazide or aminooxy moiety of a linker can be specifically attached to this aldehyde.

[0219] In some embodiments, the linker may be bonded to the polymerase by a non-covalent bond. Such a non-covalent bond is the bond of one portion of the linker to the portion fused to the polymerase. Examples of such bonding methods include fusing the polymerase to streptavidin, which can bind to the biotin portion of the linker, or fusing the polymerase to anti-digoxigenin, which can bind to the digoxigenin portion of the linker. In some embodiments, the linker's attachment to the polymerase can be brought about by site-directed conjugation, which can be easily reversed (e.g., an ortho-pyridyl disulfide (OPSS) group, which forms a disulfide bond with cysteine, which can be cleaved using a reducing agent (e.g., using TCEP)). Other binding chemical groups, on the other hand, form permanent bonds.

[0220] In any embodiment, as will be apparent to those skilled in the art, the polymerase may be mutated to specifically and reliably bind anchoring nucleotides to particular positions on the polymerase. For example, with sulfhydryl-specific binding chemical groups such as maleimide or ortho-pyridyl disulfide, accessible cysteine ​​residues in the wild-type polymerase can be mutated to non-cysteine ​​residues to prevent denaturation at such positions. In this "reactive cysteine-free" background, cysteine ​​residues can be introduced by mutation to the desired binding sites. Such mutations are selective and do not interfere with polymerase activity.

[0221] In some embodiments, the linker is specifically bound to an amino acid of the polymerase. In such cases, it is preferable to bind the linker to an amino acid at a position that can be mutated without loss of polymerase activity (e.g., positions 180, 188, 253, or 302 of mouse TdT (same numbering as in crystal structure PDB ID: 4127)). To avoid interfering with catalytic activity, it is preferable not to bind the linker to an amino acid involved in the catalytic activity of the polymerase. Residues known to be involved in catalytic activity, and methods for determining whether a residue is involved in catalytic activity (e.g., by site-directed mutagenesis), are obvious to those skilled in the art and are outlined in the 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)).

[0222] Other strategies for site-specifically attaching synthetic groups to proteins are obvious to those skilled in the art and are outlined in the literature (e.g., Stephanopoulos & Francis Nat. Chem. Biol. 7, (2011) 876-884).

[0223] In any embodiment, the polymerase may be a template-independent polymerase (i.e., a terminal deoxynucleotidyl transferase or a DNA nucleotidyl exotransferase). These terms are used synonymously to refer to enzymes having activity 2.7.7.31, using IUBMB nomenclature. The enzymes described in detail are Bolum, F. Deoxynucleotide-polymerizing enzymes of calf thymus gland. V. Homogeneous terminal deoxynucleotidyl transferase. J. Biol. Chem. 246 (1971) 909-916; Gottesman, M. and Canellakis, ES The terminal nucleotidyltransferases of calf thymus nuclei. J. Biol. Chem. 241 (1966) 4339-4352; and Krakow, JS, Coutsogeorgopoulos, C. and Canellakis, ESS studies on the incorporation of deoxyribonucleic acid. Biochim. Biophys. Acta 55 (1962) 639-650.

[0224] In some embodiments, the polymerase is mutated to improve the addition of modified nucleotides for use with free RTdNTPs.

[0225] Any polymerase capable of extending polynucleotides, any polymerase capable of incorporating nucleotides into polynucleotides, or any polymerase capable of incorporating nucleotide analogs into polynucleotides may be used in the complexes and methods described herein. In some embodiments, the polynucleotides are single-stranded. In some embodiments, the polynucleotides are double-stranded. In some embodiments, the polynucleotides are immobilized on a solid support.

[0226] Examples of DNA polymerases include polA, polB, polC, polD, polY, polX, reverse transcriptase (RT), and high-fidelity polymerases. In some cases, the polymerase is a modified polymerase. In some embodiments, the polymerases are 29, B103, GA-1, PZA, 15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, L17, ThermoSequenase®, 9°Nm®, Therminator® DNA polymerase, Tne, Tma, Tfl, Tth, TIi, Stoffel fragment, Vent® and Deep Vent® DNA polymerase, KOD DNA polymerase, Tgo, JDF-3, Pfu, Taq, T7 DNA polymerase, T7 RNA polymerase, PGB-D, UlTma DNA polymerase, Escherichia coli DNA polymerase I, Escherichia coli DNA polymerase III, Archaeon DP1I / DP2 DNA polymerase II, 9°N DNA polymerase, and Taq. This includes DNA polymerase, Phusion® DNA polymerase, Pfu DNA polymerase, SP6 RNA polymerase, RB69 DNA polymerase, avian myeloblastosis virus (AMV) reverse transcriptase, Moloney's mouse leukemia virus (MMLV) reverse transcriptase, SuperScript® II reverse transcriptase, and SuperScript® III reverse transcriptase.

[0227] In some embodiments, the polymerase is DNA polymerase 1 Krenow fragment, Vent polymerase, Phusion® DNA polymerase, KOD DNA polymerase, Taq polymerase, T7 DNA polymerase, T7 RNA polymerase, Therminator® DNA polymerase, POLB polymerase, SP6 RNA polymerase, Escherichia coli DNA polymerase I, Escherichia coli DNA polymerase III, avian myeloblastosis virus (AMV) reverse transcriptase, Moloney's mouse leukemia virus (MMLV) reverse transcriptase, SuperScript® II reverse transcriptase, or SuperScript® III reverse transcriptase.

[0228] The polymerase molecule used in the methods described herein may be polymerase theta, DNA polymerase, or any enzyme capable of elongating a nucleotide chain. In some embodiments, the polymerase is tri29. In some embodiments, the polymerase is a protein having a pocket that functions around a terminal phosphate group (e.g., a triphosphate group). In some embodiments, the methods described herein synthesize a given polynucleotide using TdT having one, two, three, four, five, six, seven, eight, nine, or ten amino acid mutations. In some embodiments, the methods described herein use TdT having one, two, three, four, five, six, seven, eight, nine, or ten amino acid mutations for surface-accessible amino acid residues. In some embodiments, the TdT used is a variant of TdT. In some embodiments, the variant of TdT includes a cysteine ​​mutation. In some embodiments, the polymerase is mutated to improve the addition of modified nucleotides bound to the polymerase forming the complex. In some cases, mutant TdT has at least 70%, 80%, 90%, or 95% sequence identity with wild-type TdT.

[0229] In some embodiments, the method described herein synthesizes a given polynucleotide using a polymerase theta having one, two, three, four, five, six, seven, eight, nine, or ten amino acid mutations. In some embodiments, the method described herein uses a polymerase theta having one, two, three, four, five, six, seven, eight, nine, or ten amino acid mutations for surface-accessible amino acid residues. In some embodiments, the polymerase theta used is a variant of polymerase theta. In some examples, the mutant polymerase theta has at least 70%, 80%, 90%, or 95% sequence identity with wild-type polymerase theta. In some embodiments, the polymerase theta is encoded by POLQ.

[0230] The enzymes described herein (e.g., TdT) include, in some embodiments, one or more non-natural amino acids. In some examples, the non-natural amino acids have lysine analogs, aromatic side chains, azide groups, alkyne groups, or aldehyde or ketone groups. In some cases, the non-natural amino acids do not include aromatic side chains. In some embodiments, the non-natural amino acids are N6-azidoethoxy-carbonyl-L-lysine (AzK), N6-propargyethoxy-carbonyl-L-lysine (PraK), N6-(propargyloxy)-carbonyl-L-lysine (PrK), p-azidophenylalanine, BCN-L-lysine, norbornenyllysine, TCO-lysine, methyltetrazinyllysine, 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-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-f Phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl Selected from N6-(((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)ceranyl)propanoic acid, 2-amino-3-(phenylceranyl)propanoic acid, selenocysteine, N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, and N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.

[0231] In some embodiments, the polymerase is a fusion protein. In some embodiments of the method, the fusion protein includes a maltose-binding protein (MBP). In some embodiments, TdT is fused to another enzyme, such as a helicase.

[0232] In some embodiments, the polymerase includes a template-independent polymerase. In some embodiments, the polymerase includes a Pol-X family polymerase. In some embodiments, the polymerase includes terminal deoxynucleotidyltransferase (TdT) or a variant thereof. In some embodiments, the template-independent polymerase includes TdT or a variant thereof. In some embodiments, TdT or a variant thereof contains a sequence having 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 with SEQ ID NO: 1. In some embodiments of the method, TdT contains the same sequence as SEQ ID NO: 1. In some embodiments, the TdT variant has one or more amino acid substitutions, amino acid insertions, or amino acid deletions with respect to SEQ ID NO: 1.

[0233] Terminal deoxynucleotidyltransferase (TdT): MGGRDIVDGSEFSPSPVPGSQNVPAPAVKKISQYACQRRTTLNNYNQLFTDALDILAENDELRENEGSALAFMRASSVLKSLPFPITSMKDTEGIPSLGDKVKSIIEGIIEDGESSEAKAVLNDERYKSFKLFTSVFGVGLKTAEKWFRMGFRTLSKIQSDKSLRFTQMQKAGFLYYEDLVSCVNRPEAEAVSMLVKEAVVTFLPDALVTMTGGFRRGKMTGHDVDFLITSPEATEDEEQQLLHKVTDFWKQQGLLLYADILESTFEKFKQPSRKVDALDHFQKCFLILKLDHGRVHSEKSGQQEGKGWKAIRVDLVMSPYDRRAFALLGWTGSRQFERDLRRYATHERKMMLDNHALYDRTKRVFLEAESEEEIFAHLGLDYIEPWERNA (Sequence ID 1)

[0234] In some embodiments, a template-independent polymerase having the activity shown in EC classification 2.7.7.31 is used. In some embodiments, the template-independent polymerase is a deoxynucleotidyl transferase or a DNA nucleotidyl exotransferase. Such enzymes are described in particular in Bollum, FJ Deoxynucleotide-polymerizing enzymes of calf thymus gland. V. Homogeneous terminal deoxynucleotidyl transferase. J. Biol. Chem. 246 (1971) 909-916; Gottesman, M. and Canellakis, ES The terminal nucleotidyltransferases of calf thymus nuclei. J. Biol. Chem. 241 (1966) 4339-4352; and Krakow, JS, Coutsogeorgopoulos, C. and Canellakis, ES Studies on the incorporation of deoxyribonucleic acid. Biochim. Biophys. Acta 55 (1962) 639-650.

[0235] Further polymerases capable of extending single-stranded nucleic acids in the absence of a usable 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 McElinny et al., Molecular cell 19.3(2005):357-366), or polymerases in which template-independent activity is induced by, for example, the insertion of template-independent polymerase components (Juarez et al., Nucleic acids Research 34.16(2006):4572-4582).

[0236] For other DNA synthesis applications, the polymerase can be a template-dependent polymerase, i.e., a DNA-dependent DNA polymerase (for example, an enzyme with activity 2.7.7.7 under IUBMB nomenclature) or an RNA-dependent DNA polymerase. Descriptions of such enzymes can be found in Richardson, A. Enzymatic synthesis of deoxyribonucleic acid. XIV. Further purification and properties of deoxyribonucleic acid polymerase of Escherichia coli. J. Biol. Chem. 239 (1964) 222-232, Schachman, A. Enzymatic synthesis of deoxyribonucleic acid. VIL Synthesis of a polymer of deoxyadenylate and deoxythymidylate. J. Biol. Chem. 235 (1960) 3242-3249, and Zimmerman, BK Purification and properties of deoxyribonucleic acid polymerase from Micrococcus lysodeikticus. J. Biol. Chem. 241 (1966) 2035-2041.

[0237] In some embodiments, the polymerase includes RNA polymerase. In such embodiments, RNA-specific nucleotidyltransferases (e.g., particularly E. coli poly(A) polymerase (IUBMB EC2.7.7.19) or poly(U) polymerase) can be used. RNA nucleotidyltransferases can be modified (e.g., single point mutations) that affect substrate specificity for particular rNTPs (Lunde et al., Nucleic acids research 40.19(2012):9815-9824). In some embodiments, a very short tethering chain can be used between the RNA nucleotidyltransferase and the ribonucleotide to induce a high effective concentration of the nucleotide, thereby promoting the incorporation of rNTPs that are not native substrates of the nucleotidyltransferase.

[0238] nucleotide In some methods, the nucleotide is ribose polyphosphate. In some methods, ribose polyphosphate is selected from the group consisting of ribose triphosphate, ribose tetraphosphate, ribose pentaphosphate, and ribose hexaphosphate. In some methods, ribose polyphosphate is ribose triphosphate. In some methods, ribose polyphosphate is ribose hexaphosphate. In some methods, ribose polyphosphate is ribose pentaphosphate. In some methods, ribose polyphosphate is ribose tetraphosphate.

[0239] In some cases, the nucleotide is deoxyribose polyphosphate. In some cases, deoxyribose polyphosphate is selected from the group consisting of deoxyribose triphosphate, deoxyribose tetraphosphate, deoxyribose pentaphosphate, and deoxyribose hexaphosphate. In some cases, deoxyribose polyphosphate is ribose triphosphate. In some cases, deoxyribose polyphosphate is deoxyribose hexaphosphate. In some cases, deoxyribose polyphosphate is deoxyribose pentaphosphate. In some cases, deoxyribose polyphosphate is deoxyribose tetraphosphate.

[0240] The term "nucleotide" and related terms refer to a molecule comprising an aromatic base, a pentose sugar (e.g., ribose or deoxyribose), and at least one phosphate group. The use of the term is consistent for both standard and non-standard nucleotides. In some embodiments, phosphate includes monophosphate, diphosphate, or triphosphate, or corresponding phosphate analogs.

[0241] Nucleotides (and nucleosides) typically consist of heterocyclic bases having substituted or unsubstituted nitrogen-containing parent heteroaromatic rings that are normally present in nucleic acids. Such nucleic acids include naturally occurring variants, substituted variants, modified variants, or engineered variants, or analogs thereof. Examples of bases include purines and pyrimidines (e.g., 2-aminopurine, 2,6-diaminopurine, adenine (A), etenoadenine, N6-Δ2-isopentenyladenine (6iA), N6-Δ2-isopentenyl-2-methylthioadenine (2ms6iA), N6-methyladenine, guanine (G), isoguanine, N2-dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and O6-methylguanine, 7-deazapurines (e.g., 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G)), pyrimidines (e.g., cytosine (C)) Examples of bases include, but are not limited to, 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O4-methylthymine, uracil (U), 4-thiouracil (4sU), and 5,6-dihydrouracil (dihydrouracil, D)), indoles (e.g., nitroindole and 4-methylindole), pyrroles (e.g., nitropyrrole), nebularin, inosine, hydroxymethylcytosine, 5-methicytosine, bases (Y), and methylated base moieties, glycosylated base moieties, and acylated base moieties. Further exemplary bases are listed in Fasman, 1989, in “Practical Handbook of Biochemistry and Molecular Biology”, pp. 385-394, CRC Press, Boca Raton, Fla.

[0242] Nucleotides (and nucleosides) typically contain a sugar moiety, such as a carbocyclic moiety (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48), an acyclic moiety (Martinez, et al., 1999 Nucleic Acids Research 27: 1271-1274, Martinez, et al., 1997 Bioorganic & Medicinal Chemistry Letters 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. Patent No. 5,558,991). The sugar portion includes ribosyl, 2'-deoxyribosyl, 3'-deoxyribosyl, 2',3'-dideoxyribosyl, 2',3'-didehydrodideoxyribosyl, 2'-alkoxyribosyl, 2'-azidoribosyl, 2'-aminoribosyl, 2'-fluororibosyl, 2'-mercaptriboxyl, 2'-alkylthioribosyl, 3'-alkoxyribosyl, 3'-azidoribosyl, 3'-aminoribosyl, 3'-fluororibosyl, 3'-mercaptriboxyl, carbocyclic 3'-alkylthioribosyl, acyclic sugars, or other modified sugars.

[0243] In some embodiments, the nucleotide comprises a chain of one, two, or three phosphorus atoms, where the chain is typically bonded to the 5' carbon of the sugar moiety via an ester bond or a phosphoramide bond. In some embodiments, the nucleotide is an analog having a phosphorus chain, in which the phosphorus atom is linked together with intervening O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atom in the chain has a substituted side group containing O, S, or BH3. In some embodiments, the chain has a phosphate group substituted with analogs such as a phosphoramidate group, a phosphorothioate group, a phosphorodithioate group, and an O-methylphosphoramidite group.

[0244] In some embodiments, the polymerase of the complex may be covalently bonded to an oligonucleotide or nucleotide via a nucleotide base. For example, the polymerase may be bonded to the nucleotide or oligonucleotide via a linker moiety at the C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base.

[0245] Furthermore, the nucleotides used in this disclosure may include natural or non-natural bases. In this regard, natural deoxyribonucleic acid may have one or more bases selected from the group consisting of adenine, thymine, cytosine, or guanine, and ribonucleic acid may have one or more bases selected from the group consisting of uracil, adenine, cytosine, or guanine. Exemplary non-natural bases that can be included in nucleic acids, regardless of whether they have a natural skeletal or analogous structure, include inosine, xatanine, hypoxatanine, isocytosine, isoguanine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiolyracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, and 6-azocytosine. Examples include, but are not limited to, zothymine, 5-uracil, 4-thiouracil, 8-haloadenine or 8-haloguanine, 8-aminoadenine or 8-aminoguanine, 8-thioladenine or 8-thiolguanine, 8-thioalkyladenine or 8-thioalkylguanine, 8-hydroxyladenine or 8-hydroxylguanine, 5-halosubstituted uracil or 5-halosubstituted cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0246] In some embodiments, the phosphorylated nucleoside (e.g., nucleotide) tethered to the polymerase is a nucleoside having at least one phosphate group. In some embodiments, the nucleoside has at least one, two, three, four, five, six, seven, eight, nine, or more than nine phosphate groups. In some embodiments, the nucleoside has at least three phosphate groups. In some embodiments, the phosphorylated nucleoside is adenosine, cytidine, uridine, or guanosine, each of which has at least one phosphate group. In some embodiments, the phosphorylated nucleoside is a deoxynucleoside having at least one phosphate group. In some embodiments, the phosphorylated nucleoside is a deoxynucleoside having at least three phosphate groups. In some embodiments, the deoxynucleoside has at least one, two, three, four, five, six, seven, eight, nine, or more than nine phosphate groups. In some embodiments, the phosphorylated nucleoside is deoxyadenosine, deoxycytidine, deoxythymidine, or deoxyguanosine, each having at least one phosphate group. In some embodiments, the phosphorylated nucleoside is nucleoside triphosphate such as dNTP. In some embodiments, the phosphorylated nucleoside is nucleoside tetraphosphate, nucleoside pentaphosphate, nucleoside hexaphosphate, nucleoside heptaphosphate, nucleoside octaphosphate, or nucleoside nonaphosphate. In some embodiments, the phosphorylated nucleoside is nucleoside hexaphosphate. In some embodiments, the phosphorylated nucleoside is nucleoside triphosphate.In some embodiments, the phosphorylated nucleoside is selected from the group consisting of deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine 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.

[0247] In some embodiments, the nucleotide analogs described herein have a reversible terminator group (e.g., an O-azidomethyl group or an O-NH2 group) at the 3' position of the sugar, or a (alpha-tert-butyl-2-nitrobenzyl)oxymethyl group at the 5 position of the pyrimidine or the 7 position of the 7-deazaprine (see, for example, Chen et al., Genomics, Proteomics & Bioinformatics 2013 11:34-40 for an overview). In such embodiments, the nucleotide analogs, once incorporated into the nucleic acid, inhibit or interfere with further elongation and control the termination of synthesis. In some embodiments, the RTdNTP-polymerase complex used as part of the complex brings about termination independently of shielding effects, for example, when the 3'-modified RTdNTP is tethered to the polymerase, and the length of the linker used may exceed 100 Å or 200 Å.

[0248] Linker In the complex, the linker is considered to be at least the atom that links the nucleotide to the polymerase. In some embodiments, the linker includes an atom that links the base, sugar, or α-phosphate of the nucleotide to the polymerase. In some embodiments, the polymerase and the nucleotide are covalently bonded, and the distance between the binding atom of the nucleotide and the polymerase to which it binds can be in the range of 4 to 100 Å (e.g., 15 to 40 Å or 20 to 30 Å), although this distance may vary depending on the position where the nucleoside triphosphate is tethered. In some embodiments, the linker can be a PEG or polypeptide linker, but again, there is considerable freedom in the type of linker used. In some embodiments, it is desirable that the linker is bonded to an atom in the nucleotide base that does not participate in base pairing. In such embodiments, the linker is thought to be located at least on an atom that links the Ca atom of the polymerase backbone to any atom of a monocyclic or polycyclic system (e.g., pyrimidine or purine or 7-deazapurine or 8-aza-7-deazapurine) bonded to the Γ position of the sugar. In other embodiments, the linker is preferably bonded to an atom involved in base pairing at the base of the nucleotide. In other embodiments, the linker is preferably bonded to the sugar or oc-phosphate of the nucleotide. In all embodiments, the linker used is preferably long enough so that the nucleoside triphosphate can access the active site of the polymerase (to which the nucleoside triphosphate is tethered). As will be described in more detail below, the polymerase of the complex can catalyze the addition of a nucleotide that binds to the 3' end of a nucleic acid.

[0249] As mentioned above, linkers can bind to various positions on nucleotides and can be cleaved using various methods. Examples of such methods are listed below, but are not limited to them.

[0250] In some embodiments, the linker can be cleaved by exposure to a reducing agent (e.g., dithiothreitol (DTT)). For example, a linker containing a 4-(disulfanyl)butanoyloxymethyl group bonded to the 5-position of a pyrimidine or the 7-position of 7-deazaprine can be cleaved by a reducing agent (e.g., DTT) to generate a 4-mercaptobutanoyloxymethyl residue on the nucleic acid base. This residue can undergo intramolecular thiolactonization, which eliminates 2-oxothiolane, leaving a smaller hydroxymethyl residue on the nucleic acid base. An example of such a linker bonded to the 5-position of cytosine is shown below, but this strategy can be applied to any suitable nucleic acid base. TIFF2026522421000026.tif36165

[0251] In other embodiments, the linker can be cleaved by exposure to light. For example, a linker containing a (2-nitrobenzyl)oxymethyl group can be cleaved with 365 nm light, leaving a hydroxymethyl residue. This is applicable to any suitable nucleic acid base, as shown below for cytosine, for example. TIFF2026522421000027.tif46165(In the formula, for example, R''=H or R''=CH3 or R'=i-Bu)

[0252] In other embodiments, the linker may contain a 3-(((2-nitrobenzyl)oxy)carbonyl)aminopropynyl group, which can be cleaved with 365 nm light to release a nucleic acid base having a propargylamino residue group. This method can be applied to any suitable nucleic acid base as follows: TIFF2026522421000028.tif36165

[0253] In other embodiments, the linker may contain an acyloxymethyl group, which can be cleaved by a suitable esterase to release a nucleic acid base having a residual hydroxymethyl group. This is applicable to any suitable nucleic acid base, as shown below for cytosine, for example. TIFF2026522421000029.tif47165

[0254] In such embodiments, the linker may include additional atoms adjacent to the ester (included in R' above), and such additional atoms increase the activity of the esterase on the ester bond.

[0255] In other embodiments, the linker may contain an N-acyl-aminopropynyl group, which can be cleaved by a peptidase to release a nucleic acid base having a propargylamino residue group. This is applicable to any suitable nucleic acid base, as shown below for example, 5-propargylaminocytosine. TIFF2026522421000030.tif40165

[0256] In such embodiments, the linker may include additional atoms adjacent to the amide (included in R' above), and such additional atoms increase the peptidase activity toward the amide bond.

[0257] In some embodiments, the polymerase-nucleotide complex comprises a nucleotide linked to the polymerase by an enzymatically cleavable linker. In some embodiments, the polymerase-nucleotide complex comprising the enzymatically cleavable linker has the structure Nuc-L 1 -L 2 -Pol is included in the formula, where Nuc represents a nucleotide, pol represents polymerase, and L 1 -L 2 represents a linker that can be cleaved by enzymes. In some embodiments, L 1 L is a nucleotide 2 This represents a region of the linker that can be cleaved by an enzyme linked to it, L 2 represents the cleavable portion of the linker that can be cleaved by the enzyme. In some embodiments, L 2 L 2 This also includes the part for connecting to Pol.

[0258] In some embodiments, the linker, which can be cleaved by enzymes, includes an amino acid ester moiety. In some embodiments, L 2 However, it contains an amino acid ester moiety. In some embodiments, the ester group of the amino acid ester moiety can be cleaved by a protease having esterase activity. 2 The amino acid esters are L 1 It is coupled to, which can also be called a spacer, or L 2 It can also be called a residual nucleotide group after ester cleavage. In some embodiments, L 2 It contains a binding chemical group for polymerase binding. In some embodiments, it further contains L 2 It contains further amino acids that bind to the amine of the amino acid ester and become a substrate for the protease. In some embodiments, in order to prevent spontaneous cleavage while retaining the ability to act as a suitable substrate for the esterase activity of a protease having esterase activity, L 2 It has been optimized for ester stability.

[0259] In some embodiments, the amino acid ester has one or more substitutions at the alpha carbon (e.g., addition of an aliphatic substituent or a bulky substituent). In some embodiments, the amino acid ester is Represented by TIFF2026522421000031.tif32165, In the formula, R 1 and R 1’ Each of these can be substituted independently of the others. 1-3 They may be selected from alkyls and halogens, or together with the atoms to which they are bonded, they may form a substituted C3-C7 carbocyclic ring.

[0260] Exemplary L(s) having different substituents on the alpha carbon of an amino acid ester (for example, to improve the stability of the ester) 2 The linker structure is shown below (L 3 L is bound to polymerase.2 (Represents part of the linker). TIFF2026522421000032.tif161165 In some embodiments, L 2 It comprises an amino acid ester adjacent to one or more amino acid residues. In some embodiments, one or more amino acid residues are bonded to the amine group of the amino acid ester.

[0261] In some embodiments, L 2 This includes or consists of the following: TIFF2026522421000033.tif27165 formula, R 1 and R1 ’ Each of these is independently hydrogen or a substituted C 1-3 They are selected from alkyl groups, or together with the atoms to which they are bonded, they form a substituted C3-C7 carboncyclic ring. Each R 3 is hydrogen, C 1-6 Alkyl, benzyl, -OH, -O(C 1-6 A optionally substituted group independently selected from alkyl and -CN, Each R c C is hydrogen or may be substituted. 1-6 It is alkyl, n is 1, 2, or 3.

[0262] In some embodiments, one or more amino acids bonded to the amine of the amino acid ester include L-isomers or D-isomers of the amino acid residue. The term “natural 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-” indicates an amino acid having a “D” (dextrorotatory) configuration as opposed to the configuration of a natural (L-) amino acid. The amino acids described herein can be purchased commercially (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art. In some embodiments, amino acids having unnatural or artificial side chains are bonded to the amine of the amino acid ester.

[0263] Linker's L 2 By selecting one or more amino acids that bind to the amino acid ester contained in the portion, for example, the binding and ester cleavage of proteases can be optimized. A combinatorial library can be generated to test the optimal cleavage activity, and amino acids can be selected based on existing known peptide sequence targets for proteases. Proteases with esterase activity can recognize the peptide portion of the linker, L 2 The ester group of the amino acid ester is hydrolyzed, resulting in the removal of polymerase bound to the nucleotide via a linker, as disclosed herein.

[0264] If necessary, a spacer can be used between the nucleotide and the linker, or between the linker and the label. L for protease / esterase 2 Spacers of different lengths can be used to increase availability and enhance the efficiency and fidelity of polymerase. Exemplary spacers include, for example, polyethylene glycol or other suitable spacers.

[0265] L containing an amino acid ester bonded to one or more amino acid residues 2 Examples of linkers with a structure are shown below, "L 3 " is L that can bind to polymerase 2 This represents the part of the linker that can be cut. TIFF2026522421000034.tif207165TIFF2026522421000035.tif89165

[0266] There is considerable freedom in the type of linker used in the region of the linker not related to enzymatic cleavage disclosed herein. Examples of suitable linker structures include, but are not limited to, carbon chain linkers (e.g., C6, C12, C18, C24, etc.), peptide linkers (e.g., polyglycine or polyalanine in the range of about 1 residue to about 1,000 residues), or polyether linkers (e.g., PEG, PPG, PAG, PTMG in the range of about 1 polyether unit to about 1,000 polyether units).

[0267] In some embodiments, the linker is a chain of atoms selected from C, N, O, S, Si, and P, preferably a chain of atoms having 0 to 500 atoms, L 1 Nuc and L 2 Covalently bonded to L 2 It is covalently bonded to Pol. 1 Atoms or L used in the formation 2 (For example, the above L 3 The atoms contained in ) can be combined in any chemically valid manner, for example, to form alkylenes, alkenylenes, and alkynylenes, and to form carbamates, carbonates, ethers, polyoxyalkylenes, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids, peptides, acyloxylamines, hydroxamic acids, or any combination thereof.

[0268] In some embodiments, the linker comprises, in different embodiments, one or more carbon atoms, zero or one or more nitrogen atoms, zero or one or more sulfur atoms, or a combination thereof. In some embodiments, the linker is 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, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000 pieces, approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 ,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,9 9, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, at least 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, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000 pieces, at least Approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 2 4, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 4 5, 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, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000 The maximum number of pieces is 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 ,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,8 5, 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, 3000, 4000, 5000, 6000, 7000, 8000, 9000,10,000 pieces, or up to approximately 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 , including 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, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or a number or range between any two of these values, containing one or more carbon atoms, one or more oxygen atoms, one or more nitrogen atoms, one or more sulfur atoms, or combinations thereof.

[0269] In some embodiments, the linker comprises a polymer such as a homopolymer or heteropolymer. In some embodiments, the linker comprises multiple repeating units. In some embodiments, the multiple repeating units comprise identical repeating units. In some embodiments, the multiple repeating units comprise two or more different repeating units. The multiple repeating units may include polyethers (e.g., paraformaldehyde, polyethylene glycol (PEG), polypropylene glycol (PPG), polyalkylene glycol (PAG), polytetramethylene glycol (PTMG), or combinations thereof). For example, the multiple repeating units may include PEGix, PEG23, PEG24, or combinations thereof. The multiple repeating units may include polyalkylenes (e.g., polyethylene, polypropene, polybutene, or combinations thereof). In some embodiments, one of the multiple repeating units does not contain an aromatic group. In some embodiments, one of the multiple repeating units contains one or more aromatic groups.

[0270] In some embodiments, the linker includes any number of basic chemical initiation blocks. For example, the linker includes linear or branched alkyl, alkenyl, or alkynyl chains, or a combination thereof, where such chains are used between the nucleotide and polymerase, nucleotide and L 2 Between, or polymerase and L 2This provides a beneficial distance between the linker and the cleavable portion. For example, amino-alkyl linkers (e.g., amino-hexyl linkers) are used to link linkers to nucleotide analogs and generally have sufficient rigidity to maintain such distances. The longest chain of such a linker may contain 2, 3, 4, 5, 6, 7, 8, 9, 10 atoms, or 11 to 35 atoms, or 35 to 50 atoms. Linear or branched linkers may also contain heteroatoms other than carbon. Such heteroatoms include, but are not limited to, oxygen, sulfur, phosphoric acid, and nitrogen. Polyoxyethylene chains (commonly known as polyethylene glycol or PEG) are preferred linker components due to the hydrophilicity of polyoxyethylene. Inserting heteroatoms such as nitrogen and oxygen into the linker can affect the solubility and stability of the linker.

[0271] Linkers may be inherently rigid or flexible. Rigid structures include laterally rigid chemical groups (e.g., cyclic structures such as aromatic compounds, multiple chemical bonds between adjacent groups, e.g., double or triple bonds), which prevent rotation of the groups relative to each other. The resulting flexibility is provided by the linker as a whole. Therefore, the required degree of rigidity can be varied depending on the linker content or the number of bonds between the individual atoms constituting the linker. Furthermore, rigidity can be imparted by adding cyclic structures along the linker. Cyclic structures may include aromatic or non-aromatic rings. The ring can be of any size, such as 3-4 carbon atoms, 3-5 carbon atoms, or even 6 carbon atoms. The ring may also optionally contain heteroatoms (e.g., oxygen or nitrogen) and may be aromatic or non-aromatic. Furthermore, the ring may be substituted with other alkyl groups and / or substituted alkyl groups.

[0272] Linkers containing a ring or aromatic structure may include, for example, aryl alkynes and aryl amides. Another example of a linker of this disclosure is an oligopeptide linker, which may also optionally contain a ring structure within its structure.

[0273] In some embodiments, the linker is C1-C 10 The alkylene chain contains 1 to 6 methylene units, independently of -NH-, -O-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, -SS-, substituted cycloalkylenes (e.g., C3-C8, C3-C6, or C5-C6), substituted heterocycloalkylenes (e.g., 3-8 member, 3-6 member, or 5-6 member), and substituted arylenes (e.g., C6-C 10 , C 10 It may be replaced by a phenylene (or substituted or unsubstituted heteroarylene (e.g., 5-10 member, 5-9 member, or 5-6 member)).

[0274] In some embodiments, L 1 The bonds, -NH-, -O-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, -SS-, will likely be substituted onto alkylenes (e.g., C1-C 20 , C 10 -C 20 , C1-C8, C1-C6, or C1-C4), substituted heteroalkylenes (e.g., 2-20 member, 8-20 member, 2-10 member, 2-8 member, 2-6 member, or 2-4 member), substituted cycloalkylenes (e.g., C3-C8, C3-C6, or C5-C6), substituted heterocycloalkylenes (e.g., 3-8 member, 3-6 member, or 5-6 member), substituted (e.g., C6-C 10 , C 10 L 1C1-C may be substituted. 20 It is an alkylene. 1 L is a 2- to 20-membered heteroalkylene, which may be substituted. In some embodiments, L 1 is a C3-C8 cycloalkylene which may be substituted. In some embodiments, L 1 L is a 3- to 8-membered heterocycloalkylene, which may be substituted. In some embodiments, L 1 C6-C may be substituted. 10 It is arylene. In some embodiments, L 1 This is a 5-10 member heteroarylene that may be substituted.

[0275] In some embodiments, L 1 This is 1 to 6 R L It is replaced by each R L These are independently oxo, halogen, -CCI3, -CBr3, -CF3, -CI3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI2, -OCHCI2, -OCHBr2, -OCHb, -OCHF2, -N3, and alkyl (e.g., C1-C) which may be substituted. 20 , C 10 -C 20 , C1-C8, C1-C6, or C1-C4), optionally substituted heteroalkyls (e.g., 2-20 membered, 8-20 membered, 2-10 membered, 2-8 membered, 2-6 membered, or 2-4 membered), optionally substituted cycloalkyls (e.g., C3-C8, C3-C6, or C5-C6), optionally substituted heterocycloalkyls (e.g., 3-8 membered, 3-6 membered, or 5-6 membered), optionally substituted aryls (e.g., C6-C 10 , C 10Selected from the group consisting of (or phenyl) and optionally substituted heteroaryls (e.g., 5-10 member, 5-9 member, or 5-6 member).

[0276] In some embodiments, L1 includes a hydroxyl-terminated group that acts as a binding site to the nucleotide and binds to a portion of L2 during synthesis.

[0277] As described herein, in some embodiments, L1 becomes a residual group that can be cleaved by the enzyme after polymerase-nucleotide linker cleavage / removal of the L2-pol moiety.

[0278] In some embodiments, L2 contains a biobinding group suitable for binding to its polymerase.

[0279] In some embodiments, the biobinding group is an N-hydroxysuccinimide (NHS) group. In some embodiments, the biobinding group is a maleimide group. Therefore, the linker may be covalently bonded to the polymerase by the reaction of the maleimide group with a cysteine ​​residue of the polymerase.

[0280] In some embodiments, the polymerase may be operably bound to a linker moiety. Such linker moieties may include covalent or non-covalent bonds, amino acid tags (e.g., poly-amino acid tags, poly-His tags, 6His tags (SEQ ID NO: 53)), compounds (e.g., polyethylene glycol), protein-protein binding pairs (e.g., biotin-avidin), affinity couplings, capture probes, or any combination thereof. The linker moiety may be separate from the polymerase variant or may be part of the polymerase variant.

[0281] The aforementioned complex can be used in nucleic acid synthesis methods. Nucleic acid synthesis can refer to the synthesis or production of a substance that is a nucleic acid molecule (e.g., polynucleotide). Nucleic acid synthesis methods can include stepwise synthesis, where nucleotides are inserted stepwise into a nucleic acid polymer or polynucleotide. As a non-limiting example, one typical process for the stepwise synthesis of polynucleotides involves stepwise adding nucleotides to a starting molecule (e.g., the first oligonucleotide) by repeatedly adding a polymerase-nucleotide complex to the oligonucleotide under conditions suitable for covalent bonding of nucleotides to the oligonucleotide's end by the catalytic action of a polymerase. The successful incorporation of the complex's nucleotides into the oligonucleotide can be called "extension" or "extension reaction," which produces an "extension product."

[0282] In some embodiments, the method comprises incubating a nucleic acid with a first complex under conditions catalyzed by polymerase to covalently add nucleotides of the first complex to the 3' hydroxyl group of the nucleic acid, thereby producing an extension product. This reaction can be carried out using nucleic acids bound to a solid support or nucleic acids in solution (e.g., not bound to a solid support). By adding the complex to the nucleic acid, a nucleic acid having an added 3' group shielded by the linked polymerase is obtained. This prevents the subsequent addition of another nucleotide while the polymerase is bound. After extending the nucleic acid with the first desired nucleotide, the method comprises a deprotection (removal of shielding) step, in which the polymerase is released from the extension product by cleaving a cleavable bond of the linker. The polymerase is removed by cleaving the linker, and a deprotected extension product is produced. Deprotection allows for further extension of the nucleic acid, and thus such a step can be periodically repeated to produce an extension product of a given sequence. Specifically, in some embodiments, the method may further include incubating the deprotected extension product with the second complex under conditions in which a polymerase catalyzes the covalent addition of a nucleotide of the second complex to the 3' end of the extension product after deprotection.

[0283] In some embodiments, the method may include: (a) incubating a nucleic acid with a first complex under conditions catalyzed by polymerase to covalently attach a nucleotide of the first complex (i.e., a single nucleotide) to the 3' hydroxyl group of the nucleic acid to produce an elongation product; (b) releasing polymerase from the elongation product and deprotecting the elongation product by cleaving a cleavable bond of the linker; (c) incubating the deprotected elongation product with a second complex under conditions catalyzed by polymerase to covalently attach a nucleotide of the second complex to the 3' end of the elongation product to produce a second elongation product; and (d) repeating steps (b) to (c) multiple times (e.g., 2 to 100 times or more) with respect to the second elongation product to produce an elongation oligonucleotide of a predetermined sequence. Steps (b) to (c) may be repeated as many times as necessary until an elongation product of a predetermined sequence and length is synthesized. The final product can be 2 to 100 base pairs long, but theoretically, this method can be used to produce products of any length (e.g., longer than 200 base pairs or longer than 500 base pairs).

[0284] In some embodiments, the nucleic acid synthesis method described herein is carried out in a reaction buffer composition. In some embodiments, the reaction buffer composition is an aqueous solution. In some embodiments, the reaction buffer composition comprises a set of components suitable for polymerase, nucleotides, polymerase-nucleotide complexes, starting molecules, nucleic acid molecular products, and the stability of any surface or matrix on which the method disclosed herein is carried out. In some such embodiments, the reaction buffer composition comprises a set of components suitable for carrying out the catalytic step described in the nucleic acid synthesis method according to this disclosure (e.g., polymerization of polynucleotides by polymerase).

[0285] The conditions for nucleic acid synthesis can vary. For example, the number of times each step is performed in a stepwise nucleotide addition can be varied to improve the purity of the multiple products produced by the nucleic acid synthesis method described herein.

[0286] In some embodiments, the nucleic acid synthesis method according to this disclosure produces a nucleic acid molecular product (i.e., a polynucleotide product). In some embodiments, the nucleic acid molecular product (i.e., a polynucleotide product) has a desired (i.e., predetermined) sequence. The “desired” or “predetermined” sequence refers to the required polynucleotide sequence intended to be produced by the nucleic acid synthesis method. The predetermined sequence may contain any number of nucleotides, including nucleic acid bases (e.g., adenine, thymine, guanine, cytosine, and / or uracil). In some embodiments, the nucleotides are modified nucleotides (i.e., nucleotide analogs). In some embodiments, the nucleic acid bases are modified nucleic acid bases. In some embodiments, the predetermined sequence may contain one or more random nucleic acid bases at predetermined positions. Including positions that result in random nucleic acid bases may be useful, for example, for introducing random mutations into the polynucleotide product.

[0287] In some embodiments, the disclosure includes a method for synthesizing a polynucleotide, the method comprising contacting a precursor polynucleotide with a complex comprising a nucleotide covalently bonded to a polymerase via a cleavable linker, wherein the nucleotide comprises a protected nucleic acid base. In some embodiments, the method for synthesizing a polynucleotide comprises adding the nucleotide to the precursor polynucleotide, and then cleaving the cleavable linker. In some embodiments, the method for synthesizing a polynucleotide comprises repeating the contact step, addition step, and optionally cleavage step described herein one or more times. In some embodiments, the removal of one or more protecting groups as described herein comprises contacting the polynucleotide with an enzyme capable of removing one or more protecting groups from a protected nucleic acid base. In some embodiments, the method for synthesizing a polypeptide comprises contacting the polynucleotide with two or more enzymes capable of removing one or more protecting groups from a protected nucleic acid base.

[0288] In some embodiments, the synthesis of polynucleotides involves stepwise adding nucleotides to a starting molecule (e.g., the first oligonucleotide) by repeating the steps of conferring a polymerase-nucleotide complex to an oligonucleotide, catalyzing the nucleotide with polymerase to attach the nucleotide to the 3' end of the oligonucleotide, and cleaving the polymerase from the added nucleotide. These steps can be repeated until the desired polynucleotide is synthesized. As described herein, using nucleotides containing protected nucleic acid bases during polynucleotide synthesis helps improve the efficiency and precision of the synthesis by suppressing the formation of secondary structures that may interfere with the addition of input nucleotides by polymerase during synthesis.

[0289] While synthesis can be completed entirely using protected nucleotides, synthesis using a combination of unmodified and protected nucleotides can also be effectively used to improve polynucleotide synthesis. In some embodiments, only one of the four nucleotides to be added (e.g., from G or T) is protected during synthesis. In some embodiments, protected nucleotides are added only to target sites where secondary or tertiary structures that may interfere with synthesis are predicted. Such structures can be predicted in various ways based on the presence of complementary DNA regions, and tools exist for each, e.g., the NUPACK algorithm (http: / / www.nuack.org / home / model). Therefore, in some embodiments, only one or two protected nucleotides can be used for the synthesis of complete polynucleotides with improved synthesis. In some embodiments, about 5%, 10%, 20%, 30%, 50%, substantially all, or 100% of a particular nucleotide are incorporated into the polynucleotide in their protected form. In some embodiments, less than 5%, less than 10%, less than 20%, less than 30%, or less than 50% of a particular nucleotide is incorporated into the polynucleotide in its protected form. In some embodiments, more than 5%, more than 10%, more than 20%, more than 30%, or more than 50% of a particular nucleotide is incorporated into the polynucleotide in its protected form. In some embodiments, only protected guanine nucleotides are used in the nucleotide synthesis reaction. Removal of protecting groups at the terminal positions of nucleic acids may be more difficult than removal from internal positions of DNA. Therefore, in some embodiments, nucleotide synthesis is carried out such that the last and first positions 1, 2, or 3 of the synthesized nucleic acid do not contain protected nucleotides.

[0290] The nucleic acid molecular products or polynucleotide products produced by the methods described herein may include a plurality of products. In some embodiments, the plurality of products include nucleic acid molecules having the desired (i.e., predetermined) sequence. In some embodiments, the plurality of products include nucleic acid molecules having sequences other than the desired sequence. In some embodiments, the plurality of products include nucleic acid molecular products having the desired sequence and nucleic acid molecular products not having the desired sequence. The "purity" of the plurality of products may refer to the ratio of the abundance of nucleic acid molecular products having the desired sequence to the abundance of nucleic acid molecular products not having the desired sequence. The purity of the product can be evaluated by a variety of methods known in the art for sequencing nucleic acids. Any suitable nucleic acid sequencing method can be used. For example, the product can be evaluated by Sanger sequencing, next-generation sequencing (e.g., Illumina sequencing), or long-read sequencing (e.g., small molecule real-time sequencing (SMRT) and nanopore sequencing), for example.

[0291] In some embodiments, the nucleic acid synthesis method according to this disclosure produces a product having a purity of about 10% to about 99.99%. In some embodiments, the nucleic acid synthesis method produces a product having a purity of at least 10%. In some embodiments, the nucleic acid synthesis method produces a product having a purity of at least 10%. In some embodiments, the nucleic acid synthesis method produces a product having a purity of at least 20%. In some embodiments, the nucleic acid synthesis method produces a product having a purity of at least 30%. In some embodiments, the nucleic acid synthesis method produces a product having a purity of at least 40%. In some embodiments, the nucleic acid synthesis method produces a product having a purity of at least 50%. In some embodiments, the nucleic acid synthesis method produces a product having a purity of at least 60%. In some embodiments, the nucleic acid synthesis method produces a product having a purity of at least 70%. In some embodiments, the nucleic acid synthesis method produces a product having a purity of at least 80%. In some embodiments, the nucleic acid synthesis method produces a product having a purity of at least 90%. In some embodiments, the nucleic acid synthesis method produces a product having a purity of at least 95%. In some embodiments, the nucleic acid synthesis method produces a product having a purity of at least 99%.

[0292] In any of the embodiments summarized above, the nucleoside triphosphate can be deoxyribonucleoside triphosphate or ribonucleoside triphosphate. In some embodiments, the complex may include RNA polymerase linked to ribonucleoside triphosphate. In such embodiments, the nucleotide added to the nucleic acid may be a ribonucleotide. In other embodiments, the complex may include DNA polymerase linked to deoxyribonucleoside triphosphate. In such embodiments, the nucleotide added to the nucleic acid may be a deoxyribonucleotide.

[0293] In some embodiments, the nucleotide is a nucleotide analog. In some embodiments, the nucleotide analog is a reversible terminator. Reversible terminators are known in the art for use in nucleic acid synthesis. The use of reversible terminators in nucleic acid synthesis has been previously reported. See, for example, WO2021 / 122539A1, WO2018 / 215803A1, WO2021 / 094251A1, and WO2020 / 081985A1.

[0294] In some embodiments, the nucleotide may include a reversible terminator (RTdNTP), and the deprotection step of the method further includes removing a protecting group (e.g., removing a terminator group) from the added nucleotide to produce a deprotected extension product. Deprotection allows for further extension of the nucleic acid, and thus such a step can be periodically repeated to produce an extension product of a given sequence.

[0295] A sequencing method is also provided. Such a method includes incubating a double helix containing primers and a template with a composition containing a series of complexes (where the complexes correspond to G, A, T, and C and are identifiablely labeled (e.g., fluorescently labeled)); detecting which nucleotides were attached to the primers by detecting the labels tethered to the polymerase to which the nucleotides were attached; deprotecting the extension product by cleaving the linker; and repeating the steps of incubation, detection, and deprotection to obtain the sequence of at least a portion of the template.

[0296] This disclosure describes an enzymatic method for polynucleotide synthesis, which uses a polymerase-nucleotide complex to repeatedly add a single nucleotide per cycle to the 3' hydroxyl end of an elongated polynucleotide chain, controlled by a nucleotide-binding polymerase. Such control is achieved by a so-called "shielding effect." Shielding represents steric hindrance, which prevents the 3' hydroxyl end extended by one complex from being accessed by another complex, while keeping the polymerase bound to the added nucleotide and further preventing the polymerase, tethered to the nucleotide at the 3' end, from accessing the nucleotide of another complex.

[0297] PCT Publication WO2017 / 223517 (in its entirety incorporated herein by reference) describes a typical process for stepwise synthesis of a given sequence using a template-independent polymerase. A nucleic acid acting as the initial substrate for extension (i.e., the "starting molecule") is incubated with a first polymerase-nucleotide complex. Once the nucleic acid is extended by the nucleotides linked by the complex, no further extension occurs as the complex executes a termination mechanism. In the second step of the process, the linker is cleaved to release the polymerase and reverse the termination mechanism, thereby enabling further extension. The extension product is then exposed to the second complex, and these two steps are repeated to extend the nucleic acid by the given sequence. Synthetic procedures using complexes containing TdT and photocleavable linkers are also described in WO2017 / 223517. As mentioned above, other methods are also available for linker binding and cleavage.

[0298] A key step in this approach to polynucleotide synthesis is deprotection, i.e., the removal of the tethered polymerase from the elongated polynucleotide, thereby making the 3' end available for continued elongation in the next synthesis cycle. For usefulness in polynucleotide synthesis, the removal of the tethered polymerase is preferably carried out at a rapid reaction rate to shorten the synthesis cycle time, while being carried out under mild conditions to prevent damage to the polynucleotide being synthesized. Furthermore, the removal of the tethered polymerase is preferably allowed to proceed until it is completely finished, and it is preferable to produce cleavage products that do not hinder the continued elongation or subsequent use of the resulting DNA synthesis product. In some embodiments, the tethered strand also enables efficient binding of the nucleotide to the polymerase, subsequently effectively positioning the nucleotide within the active site to facilitate rapid integration of the free primer into the 3' end.

[0299] In this specification, the inventors describe the design of an optimized cleavable linker used to tether polymerase to nucleotides. Such a linker is highly stable during storage and under oligo synthesis reaction conditions (before controlled linker cleavage) and can be enzymatically cleaved within a short time suitable for oligo synthesis.

[0300] This specification provides a complex comprising a polymerase and a nucleotide linked via a linker having an enzymatically cleavable bond. The polymerase portion of the complex can extend the nucleic acid using its linked nucleotide (i.e., the polymerase can catalyze the binding of the nucleotide to which it is linked to the nucleic acid), and remains linked to the extended nucleic acid via the linker until the linker is cleaved by the enzyme.

[0301] In the complex, the linker contains an atom that links the nucleotide to the polymerase. In some embodiments, the linker links the base, sugar, or α-phosphate of the nucleotide to the polymerase. In some embodiments, the linker links the terminal phosphate of the nucleotide to the polymerase. In some embodiments, the linker links the nucleotide to the C in the polymerase backbone. α Linking to an atom. In some embodiments, the polymerase and the nucleotide are linked by a covalent bond, and the distance between the bonding atom of the nucleotide and the polymerase to which it is linked can be in the range of 4 to 100 Å (e.g., 15 to 40 Å or 20 to 30 Å). However, this distance may vary depending on the position to which the nucleotide is linked. It is desirable that the linker used be long enough so that the nucleotide can access the active site of the polymerase to which it is linked. As shown in more detail below, the polymerase of the complex can catalyze the reaction to add the nucleotide to which it is linked to the 3' end of the nucleic acid.

[0302] Furthermore, the linkers intended herein have sufficient length and stability to enable efficient hydrolysis by enzymatic means. The number of carbons or atoms in the linker, which may be derivatized by other functional groups, must be long enough to allow the polymerase to cleave the nucleotides by the enzyme.

[0303] In certain embodiments, the cleavable linker contains an amino acid ester. In some embodiments, the amino acid ester is a cleavage site of the linker, thereby facilitating polymerase release upon exposure to an esterase or protease having esterase activity. The portion of the cleavable linker containing the amino acid ester is referred to herein as "L" of the linker. 2 This is called the "part". 2This can be designed and optimized for enzymatic cleavage by esterases or proteases having esterase activity. Such design and optimization can be achieved, for example, by modifying the chemical group bonded to the alpha carbon of the amino acid ester, or by modifying one or more amino acids adjacent to the amino acid ester. 2 This can be done by including it as part of something.

[0304] This specification describes polymerase-nucleotide complexes containing a cleavable linker. The cleavable linker is highly stable and can be rapidly enzymatically cleaved by a protease having esterase activity. In some embodiments, the polymerase-nucleotide complex comprises a nucleotide linked to a polymerase by an enzymatically cleavable linker. In some embodiments, the polymerase-nucleotide complex containing an enzymatically cleavable linker has the structure Nuc-L 1 -L 2 -L 3 -Polyx, where Nuc represents a nucleotide and pol represents polymerase, L 1 -L 2 -L 3 represents a linker that can be cleaved by enzymes. In some embodiments, L 1 L is a nucleotide 2 This represents a region of the linker that is linked to and can be cleaved by enzymes, L 2 L represents the cleavable portion of the linker that can be cleaved by enzymes. 3 L 2 This represents the region of the linker that connects to Pol and can be cleaved by enzymes.

[0305] In some embodiments, the linker, which can be cleaved by enzymes, includes an amino acid ester moiety. In some embodiments, L 2 However, it contains an amino acid ester moiety. In some embodiments, the ester group of the amino acid ester moiety can be cleaved by a protease having esterase activity. 2 The amino acid esters are L 1It is coupled to, which can also be called a spacer, or L 2 It can also be called a residual nucleotide group after ester cleavage. 2 L 3 It is bonded to (the rest of the linker), which has a binding chemical group for polymerase binding. In some embodiments, L 3 It may also include, or be called, a spacer. In some embodiments, it may further include L 2 It contains further amino acids that bind to the amine of the amino acid ester to become a substrate for the protease. As described herein, in order to prevent spontaneous cleavage while retaining the ability to act as a suitable substrate for the esterase activity of a protease having esterase activity, L 2 It has been optimized for ester stability.

[0306] In some embodiments, the linker is bound to the nucleic acid base of the nucleotide. In some embodiments, the linker is bound to the sugar of the nucleotide. In some embodiments, the linker is bound to the 5' phosphate group of the nucleotide, where 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 gamma phosphate, beta phosphate, delta phosphate, epsilon phosphate, zeta phosphate, etha phosphate, or theta phosphate. In some embodiments, the linker is bound to the terminal phosphate. In some embodiments, the linker of the complex can be bound to the 7th position of dGTP, or to the 5th position of dTTP or dUTP.

[0307] Further examples of tethered creotides are described, for example, in PCT Publication WO2017 / 223517, “Nucleic Acid Synthesis and Sequencing Using Tethered Nucleoside Triphosphates,” the entire document of which is referenced by reference.

[0308] In some embodiments, sulfhydryl-specific binding groups can be used to specifically conjugate nucleotides to cysteine ​​residues of polymerases. Possible sulfhydryl-specific binding groups include, but are not limited to, ortho-pyridyl disulfide (OPSS), maleimide functional groups, 3-arylpropioronitrile functional groups, arenamide functional groups, haloacetyl functional groups (e.g., iodoacetyl or bromoacetyl), alkyl halides, or perfluoroaryl groups that react well with sulfhydryls surrounded by specific amino acid sequences (Zhang, Chi, et al. Nature Chemistry 8, (2015): 120-128). Other binding groups for the specific conjugation of cysteine ​​residues are obvious to those skilled in the art or are described in relevant literature and texts (e.g., Kim, Younggyu, et al. Bioconjugate Chemistry 19.3 (2008): 786-791).

[0309] Several TdT-dNTP complexes were prepared and assayed using various cleavable linkers that specifically ligate nucleotide sites to polymerases. Using peptide bonds in the linkers yields linkers that are cleavable by proteases. Protease-mediated cleavage of the peptide bond generates amines and carboxylic acids, both of which possess a charge under typical buffer conditions for TdT activity. However, the persistence of charged functional groups on the synthesized oligonucleotides can have detrimental effects during synthesis.

[0310] In contrast, cleavage of the ester group generates an alcohol, which is a charge-neutral cleavage product. In this specification, the inventors demonstrate that nucleotides having such alcohol-containing residual groups do not interfere with oligonucleotide synthesis by the complex (see Example 2). The inventors also demonstrate that linkers containing amino acid esters can be enzymatically cleaved by proteases having esterase activity, such as proteinase K (see Example 2).

[0311] Therefore, in some embodiments, L 2 It contains an amino acid ester moiety. In some embodiments, the amino acid ester is a linker cleavage site that facilitates the release of polymerase from the nucleotide.

[0312] Furthermore, as the inventors initially acknowledged, the ester group of the glycine amino acid ester in the linker is unstable and could potentially lead to spontaneous cleavage of the complex and unwanted nucleotide insertions during oligonucleotide synthesis by the complex (see Examples 2 and 3). However, it was found that adding an aliphatic substituent or a bulky substituent to the alpha carbon of the amino acid ester significantly improved the stability of the adjacent ester (see Examples 4 and 6). Moreover, atomic substitution at the alpha carbon of the amino acid ester can affect hyperconjugation, potentially increasing or decreasing the instability of the adjacent ester, and potentially increasing or decreasing the cleavage rate by proteases with esterase activity. Therefore, by selecting a preferred substituent at the alpha carbon of the amino acid ester, a favorable balance between stability and linker cleavage rate can be achieved (see Example 6).

[0313] Therefore, in some embodiments, the amino acid ester has one or more substituents on the alpha carbon (e.g., aliphatic substituents or bulky substituents). In some embodiments, the amino acid ester is Represented by TIFF2026522421000036.tif32165, In the formula, R 1 and R 1’ Each of these can be substituted independently of the others. 1-3 They may be selected from alkyls and halogens, or together with the atoms to which they are bonded, they may form a substituted C3-C7 carbocyclic ring.

[0314] Exemplary L(s) having different substituents on the alpha carbon of an amino acid ester (for example, to improve the stability of the ester) 2 The linker structure is shown below. TIFF2026522421000037.tif161165

[0315] Furthermore, as the inventors have observed, the rate of ester group cleavage by a protease having esterase activity is the rate of L adjacent to the amino acid ester. 2 This can be improved by including one or more amino acids in the portion (see Example 5). In some embodiments, L 2 It comprises an amino acid ester adjacent to one or more amino acid residues. In some embodiments, one or more amino acid residues are bonded to the amine group of the amino acid ester.

[0316] In some embodiments, L 2 This includes or consists of the following: TIFF2026522421000038.tif27165 formula, R 1 and R1 ’ Each of these is independently hydrogen or a substituted C 1-3 They are selected from alkyl groups, or together with the atoms to which they are bonded, they form a substituted C3-C7 carboncyclic ring. Each R 3 is hydrogen, C 1-6 Alkyl, benzyl, -OH, -O(C 1-6 A optionally substituted group independently selected from alkyl and -CN, Each R c C is hydrogen or may be substituted. 1-6 It is alkyl, n is 1, 2, or 3.

[0317] In some embodiments, one or more amino acids bonded to the amine of the amino acid ester include L-isomers or D-isomers of the amino acid residue. The term “natural 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-” indicates an amino acid having a “D” (dextrorotatory) configuration as opposed to the configuration of a natural (L-) amino acid. The amino acids described herein can be purchased commercially (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art. In some embodiments, amino acids having unnatural or artificial side chains are bonded to the amine of the amino acid ester.

[0318] As described above, the present inventors have determined the composition of the linker (i.e., L 2 We found that the peptide sequence of L significantly affects the rate of protease-mediated deprotection. Therefore, 2 Various rearrangements of amino acids in a linker can result in complexes with faster addition and deprotection reaction rates. Such linkers can include variations in the types of amino acids and the number of consecutive amino acids.

[0319] Linker's L 2 By selecting one or more amino acids that bind to the amino acid ester contained in the portion, for example, the binding and ester cleavage of proteases can be optimized. A combinatorial library can be generated to test the optimal cleavage activity, and amino acids can be selected based on existing known peptide sequence targets for proteases. Proteases with esterase activity can recognize the peptide portion of the linker, L 2 The ester group of the amino acid ester is hydrolyzed, resulting in the removal of polymerase bound to the nucleotide via a linker, as disclosed herein.

[0320] Spacers can be used between the nucleotide and the linker, or between the linker and the label, as needed. Spacers of different lengths can be used to increase the availability of L2 to the protease / esterase and to increase the efficiency and fidelity of the polymerase. Exemplary spacers include, for example, polyethylene glycol or other suitable spacers.

[0321] L containing an amino acid ester bonded to one or more amino acid residues 2 Examples of linkers with a structure are shown below. TIFF2026522421000039.tif168165TIFF2026522421000040.tif130165

[0322] Regions of the linker not involved in the enzymatic cleavage disclosed herein (e.g., L 1 and L 3 There is considerable flexibility in the type of linker used. Suitable linker structures include, but are not limited to, carbon chain linkers (e.g., C6, C12, C18, C24, etc.), peptide linkers (e.g., polyglycine or polyalanine in the range of approximately 1 residue to approximately 1,000 residues), or polyether linkers (e.g., PEG, PPG, PAG, PTMG in the range of approximately 1 polyether unit to approximately 1,000 polyether units).

[0323] In some embodiments, L 1 or L 3 L is a chain of atoms selected from C, N, O, S, Si, and P, preferably a chain having 0 to 500 atoms, where L 1 Nuc and L 2 L is connected by a covalent bond. 3 L 2 L and Pol are connected by a covalent bond. 1 or L 3The atoms used to form can be combined in any chemically reasonable manner, for example, alkylenes, alkenylenes, and alkynylenes, and can form carbamates, carbonates, ethers, polyoxyalkylenes, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids, peptides, acyloxylamines, hydroxamic acids, or any combination thereof.

[0324] In some embodiments, L 1 or L 3 In different embodiments, L includes one or more carbon atoms, zero or one or more oxygen atoms, zero or one or more nitrogen atoms, zero or one or more sulfur atoms, or a combination thereof. In some embodiments, L 1 or L 3The numbers are 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 ,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,8 6, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 100 00 pieces, approximately 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 ,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,8 5, 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, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 pieces, at least 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, 4 1, 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, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, at least 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, 3 8, 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, 3000, 4000, 5000, 6000 , 7000, 8000, 9000, 10000, with a maximum of 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 ,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,7 8, 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, 3000, 4000, 5000 , 6000, 7000, 8000, 9000, 10000 pieces, or up to approximately 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 , including 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000 carbon atoms, oxygen atoms, nitrogen atoms, sulfur atoms, or combinations thereof, or numbers or ranges between any two of these values.

[0325] In some embodiments, L 1 or L 3 This includes polymers such as homopolymers or heteropolymers. In some embodiments, L 1 or L 3 The repeating units include multiple repeating units. In some embodiments, the repeating units include identical repeating units. In some embodiments, the repeating units include two or more different repeating units. The repeating units may include polyethers (e.g., paraformaldehyde, polyethylene glycol (PEG), polypropylene glycol (PPG), polyalkylene glycol (PAG), polytetramethylene glycol (PTMG), or combinations thereof). For example, the repeating units may include PEGix, PEG23, PEG24, or combinations thereof. The repeating units may include polyalkylenes (e.g., polyethylene, polypropene, polybutene, or combinations thereof). In some embodiments, one of the repeating units does not contain an aromatic group. In some embodiments, one of the repeating units contains one or more aromatic groups.

[0326] In some embodiments, L 1 or L 3 It contains any number of basic chemical initiation blocks. For example, the linker may contain linear or branched alkyl chains, alkenyl chains, or alkynyl chains, or a combination thereof, where such chains are between the nucleotide and polymerase, nucleotide and L 2 Between, or polymerase and L 2 This provides a beneficial distance between the linker and the nucleotide analogue. For example, amino-alkyl linkers (e.g., amino-hexyl linkers) are used to link linkers to nucleotide analogues and generally have sufficient rigidity to maintain such distances. The longest chain of such linkers can contain 2, 3, 4, 5, 6, 7, 8, 9, 10 atoms, or 11 to 35 atoms, or 35 to 50 atoms. Linear or branched linkers may also contain heteroatoms other than carbon. Such heteroatoms include, but are not limited to, oxygen, sulfur, phosphorus, and nitrogen. Polyoxyethylene chains (commonly known as polyethylene glycol or PEG) are preferred linker components due to the hydrophilicity of polyoxyethylene. Inserting heteroatoms such as nitrogen and oxygen into the linker can affect the solubility and stability of the linker.

[0327] L 1 or L 3Linkers containing these groups may be inherently rigid or flexible. Rigid structures include laterally rigid chemical groups (e.g., cyclic structures such as aromatic compounds, multiple chemical bonds between adjacent groups, e.g., double or triple bonds), which prevent rotation of the groups relative to each other. The resulting flexibility is provided by the linker as a whole. Therefore, the desired degree of rigidity can be varied depending on the linker content or the number of bonds between the individual atoms constituting the linker. Furthermore, rigidity can be imparted by adding cyclic structures along the linker. Cyclic structures may include aromatic or non-aromatic rings. The rings can be of any size, such as 3-4 carbon, 3-5 carbon, or even 6 carbon. The rings may also optionally contain heteroatoms (e.g., oxygen or nitrogen) and may be aromatic or non-aromatic. Furthermore, the rings may be substituted with other alkyl groups and / or substituted alkyl groups.

[0328] Linkers containing a ring or aromatic structure may include, for example, aryl alkynes and aryl amides. Another example of a linker of this disclosure is an oligopeptide linker, which may also optionally contain a ring structure within its structure.

[0329] In some embodiments, L 1 or L 3 C1-C 10 An alkylene chain, where 1 to 6 methylene units are independently -NH-, -O-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, -SS-, substituted cycloalkylenes (e.g., C3-C8, C3-C6, or C5-C6), substituted heterocycloalkylenes (e.g., 3-8 member, 3-6 member, or 5-6 member), substituted arylenes (e.g., C6-C 10 , C 10 It may be replaced by a phenylene (or substituted or unsubstituted heteroarylene (e.g., 5-10 member, 5-9 member, or 5-6 member)).

[0330] In some embodiments, L 1 or L 3 The bonds, -NH-, -O-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, -SS-, will likely be substituted onto alkylenes (e.g., C1-C 20 , C 10 -C 20 , C1-C8, C1-C6, or C1-C4), substituted heteroalkylenes (e.g., 2-20 member, 8-20 member, 2-10 member, 2-8 member, 2-6 member, or 2-4 member), substituted cycloalkylenes (e.g., C3-C8, C3-C6, or C5-C6), substituted heterocycloalkylenes (e.g., 3-8 member, 3-6 member, or 5-6 member), substituted (e.g., C6-C 10 , C 10 L 1 or L 3 C1-C may be substituted. 20 It is an alkylene. 1 or L 3 L is a 2- to 20-membered heteroalkylene, which may be substituted. In some embodiments, L 1 or L 3 is a C3-C8 cycloalkylene which may be substituted. In some embodiments, L 1 or L 3 L is a 3- to 8-membered heterocycloalkylene, which may be substituted. In some embodiments, L 1 or L 3 C6-C may be substituted. 10 It is arylene. In some embodiments, L 1 or L 3 This is a 5-10 member heteroarylene that may be substituted.

[0331] In some embodiments, L1 This is 1 to 6 R L It is replaced by L 3 This is 1 to 6 R L It is replaced by each R L These are independently oxo, halogen, -CCI3, -CBr3, -CF3, -CI3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI2, -OCHCI2, -OCHBr2, -OCHb, -OCHF2, -N3, and alkyl (e.g., C1-C) which may be substituted. 20 , C 10 -C 20 , C1-C8, C1-C6, or C1-C4), optionally substituted heteroalkyls (e.g., 2-20 membered, 8-20 membered, 2-10 membered, 2-8 membered, 2-6 membered, or 2-4 membered), optionally substituted cycloalkyls (e.g., C3-C8, C3-C6, or C5-C6), optionally substituted heterocycloalkyls (e.g., 3-8 membered, 3-6 membered, or 5-6 membered), optionally substituted aryls (e.g., C6-C 10 , C 10 Selected from the group consisting of (or phenyl) and optionally substituted heteroaryls (e.g., 5-10 member, 5-9 member, or 5-6 member).

[0332] In some embodiments, L 1 or L 3 is -(CH2CH2O) b - is. In some embodiments, L 1 or L 3 is -CCCH2(OCH2CH2) a -NHC(O)-(CH2)c(OCH2CH2) b - is. In some embodiments, L 1 or L 3 is -CHCHCH2-NHC(O)-(CH2) c (0CH2CH2)b - is. In some embodiments, L 1 or L 3 is -CCCH2-NHC(O)-(CH2)c(OCH2CH2) b - is. In some embodiments, L 1 or L 3 is -CCCH2-. Symbol a is an integer from 0 to 8. In some embodiments, a is 1. In some embodiments, a is 0. Symbol b is an integer from 0 to 8. In some embodiments, b is 1 or 2. In some embodiments, b is an integer from 2 to 8. In some embodiments, b is 1. Symbol c is an integer from 0 to 8. In some embodiments, c is 3. In some embodiments, c is 1. In some embodiments, c is 2. In some embodiments, L1 or L3 are independently substituted or unsubstituted C1-C4 alkylenes, or substituted or unsubstituted 8-20 member heteroalkylenes.

[0333] L1 acts as a binding site to the nucleotide and contains a hydroxyl terminal group that binds to a portion of L2 during synthesis.

[0334] In some embodiments, L1 becomes a residual group that can be cleaved enzymatically or chemically after polymerase-nucleotide linker cleavage / removal of the L2-L3-pol moiety.

[0335] In some embodiments, L1 may be bonded to or replaced by C 1-12 Alkylene chain, C4-C 20 Polyethylene glycol, may be substituted C 2-12 Alkenylene chain and optionally substituted C 2-12 Selected from the group consisting of alkynylene chains, where L 1 The 1 to 4 methylene units are independently -O-, -N(R b )-, -C(O)-, -S-, -S(O)-, -S(O)2-, phenylene, cyclopropylene, where each R b C is independently hydrogen or may be substituted. 1-6 It is alkyl.

[0336] In some embodiments, L1 is Includes TIFF2026522421000041.tif47165, In the formula, each R a C may be independently a halogen, hydroxyl, cyano, or substituted. 1-6 Alkyl and optionally substituted C 1-6 Selected from the group consisting of alkoxys.

[0337] In some embodiments, L 1 teeth, Selected from the group consisting of TIFF2026522421000042.tif58165.

[0338] In some embodiments, L3 includes a biobinding group suitable for binding L3 to polymerase.

[0339] In some embodiments, the biobinding group is an N-hydroxysuccinimide (NHS) group. In some embodiments, the biobinding group is a maleimide group. Therefore, the linker may be covalently bonded to the polymerase by the reaction of the maleimide group with a cysteine ​​residue of the polymerase.

[0340] In some embodiments, the polymerase may be operably bound to a linker moiety. Such linker moieties may include covalent or non-covalent bonds, amino acid tags (e.g., poly-amino acid tags, poly-His tags, 6His tags (SEQ ID NO: 53)), compounds (e.g., polyethylene glycol), protein-protein binding pairs (e.g., biotin-avidin), affinity couplings, capture probes, or any combination thereof. The linker moiety may be separate from the polymerase variant or may be part of the polymerase variant.

[0341] In some embodiments, the linker connecting the nucleotide and polymerase comprises saturated or unsaturated, substituted or unsubstituted, linear or branched carbon chains. The linker length may vary in different embodiments. The linker length may vary depending on the type of nucleotide and polymerase. In some embodiments, the linker length in an enzyme-linked nucleotide varies for each different nucleotide or nucleotide analogue. In one embodiment, the linker lengths are 19 Å, 20 Å, 21 Å, 22 Å, 23 Å, 24 Å, 25 Å, 26 Å, 27 Å, 28 Å, 29 Å, 30 Å, 31 Å, 32 Å, 33 Å, 34 Å, 35 Å, 36 Å, 37 Å, 38 Å, 39 Å, 40 Å, 41 Å, 42 Å, 43 Å, 44 Å, 45 Å, 46 Å, 47 Å, 48 Å, 49 Å, 50 Å, 51 Å, 52 Å, 53 Å, 54 Å, 55 Å, 56 Å, 57 Å, 58 Å, 59 Å, 60 Å, 61 Å, 62 Å, 63 Å, 64Å, 65Å, 66Å, 67Å, 68Å, ​​69Å, 70Å, 71Å, 72Å, 73Å, 74Å, 75Å, 76Å, 77Å, 78Å, 79Å, 80Å, 81Å, ​​82Å, 83Å, 84Å, 85Å, 86Å, 87Å, 8 8Å, 89Å, 90Å, 91Å, 92Å, 93Å, 94Å, 95Å, 96Å, 97Å, 98Å, 99Å, 100Å, 200Å, 300Å, 400Å, 500Å, 600Å, 700Å, 800Å, 900Å, 1000Å, approx. 9Å, 20Å, 21Å, 22Å, 23Å, 24Å, 25Å, 26Å, 27Å, 28Å, 29Å, 30Å, 31Å, 32Å, 33Å, 34Å, 35Å, 36Å, 37Å, 38Å, 39Å, 40Å, 41Å, 42Å, 43Å, 44Å, 45Å, 46Å, 47Å, 48Å, 49Å, 50Å, 51Å, 52Å, 53Å, 54Å, 55Å, 56Å, 57Å, 58Å, 59Å, 60Å, 61Å, 62Å, 63Å, 64Å, 65Å, 66Å, 67Å, 68Å, 69 Å, 70 Å, 71 Å, 72 Å, 73 Å, 74 Å, 75 Å, 76 Å, 77 Å, 78 Å, 79 Å, 80 Å, 81 Å, 82 ​​Å, 83 Å, 84 Å, 85 Å, 86 Å, 87 Å, 88 Å, 89 Å, 90 Å, 91 Å, 92 Å, 93 Å, 94 Å, 95 Å, 96 Å, 97 Å, 98 Å, 99 Å, 100 Å, 200 Å, 300 Å, 400 Å, 500 Å, 600 Å, 700 Å, 800 Å, 900 Å, 1000 Å, at least 19 Å, 20 Å, 21 Å, 22 Å, 23 Å,24Å, 25Å, 26Å, 27Å, 28Å, 29Å, 30Å, 31Å, 32Å, 33Å, 34Å, 35Å, 36Å, 37Å, 38Å, 39 Å, 40Å, 41Å, 42Å, 43Å, 44Å, 45Å, 46Å, 47Å, 48Å, 49Å, 50Å, 51Å, 52Å, 53Å, 54Å, 55Å, 56Å, 57Å, 58Å, 59Å, 60Å, 61Å, 62Å, 63Å, 64Å, 65Å, 66Å, 67Å, 68Å, ​​69Å, 70 Å, 71Å, 72Å, 73Å, 74Å, 75Å, 76Å, 77Å, 78Å, 79Å, 80Å, 81Å, ​​82Å, 83Å, 84Å, 85Å, 8 6 Å, 87 Å, 88 Å, 89 Å, 90 Å, 91 Å, 92 Å, 93 Å, 94 Å, 95 Å, 96 Å, 97 Å, 98 Å, 99 Å, 100 Å, 200 Å, 300 Å, 400 Å, 500 Å, 600 Å, 700 Å, 800 Å, 900 Å, 1000 Å, at least about 19 Å, 20 Å, 21 Å, 2 2Å, 23Å, 24Å, 25Å, 26Å, 27Å, 28Å, 29Å, 30Å, 31Å, 32Å, 33Å, 34Å, 35Å, 36Å, 37Å , 38Å, 39Å, 40Å, 41Å, 42Å, 43Å, 44Å, 45Å, 46Å, 47Å, 48Å, 49Å, 50Å, 51Å, 52Å, 53 Å, 54Å, 55Å, 56Å, 57Å, 58Å, 59Å, 60Å, 61Å, 62Å, 63Å, 64Å, 65Å, 66Å, 67Å, 68Å, 69Å, 70Å, 71Å, 72Å, 73Å, 74Å, 75Å, 76Å, 77Å, 78Å, 79Å, 80Å, 81Å, ​​82Å, 83Å, 84Å , 85Å, 86Å, 87Å, 88Å, 89Å, 90Å, 91Å, 92Å, 93Å, 94Å, 95Å, 96Å, 97Å, 98Å, 99Å, 100Å, 200Å, 300Å, 400Å, 500Å, 600Å, 700Å, 800Å, 900Å, 1000Å, with a maximum of 19Å, 20Å, 21 Å, 22Å, 23Å, 24Å, 25Å, 26Å, 27Å, 28Å, 29Å, 30Å, 31Å, 32Å, 33Å, 34Å, 35Å, 36Å, 37Å, 38Å, 39Å, 40Å, 41Å, 42Å, 43Å, 44Å, 45Å, 46Å, 47Å, 48Å, 49Å, 50Å, 51Å, 52Å , 53Å, 54Å, 55Å, 56Å, 57Å, 58Å, 59Å, 60Å, 61Å, 62Å, 63Å, 64Å, 65Å, 66Å, 67Å, 6 8Å, 69Å, 70Å, 71Å, 72Å, 73Å, 74Å, 75Å, 76Å, 77Å, 78Å, 79Å, 80Å, 81Å, ​​82Å, 83Å,84 Å, 85 Å, 86 Å, 87 Å, 88 Å, 89 Å, 90 Å, 91 Å, 92 Å, 93 Å, 94 Å, 95 Å, 96 Å, 97 Å, 98 Å, 99 Å, 100 Å, 200 Å, 300 Å, 400 Å, 500 Å, 600 Å, 700 Å, 800 Å, 900 Å, 1000 Å, or up to approximately 19 Å, 20 Å, 21 Å , 22Å, 23Å, 24Å, 25Å, 26Å, 27Å, 28Å, 29Å, 30Å, 31Å, 32Å, 33Å, 34Å, 35Å, 36Å, 37Å, 38 Å, 39Å, 40Å, 41Å, 42Å, 43Å, 44Å, 45Å, 46Å, 47Å, 48Å, 49Å, 50Å, 51Å, 52Å, 53Å, 54Å, 5 5Å, 56Å, 57Å, 58Å, 59Å, 60Å, 61Å, 62Å, 63Å, 64Å, 65Å, 66Å, 67Å, 68Å, ​​69Å, 70Å, 71Å, 72Å, 73Å, 74Å, 75Å, 76Å, 77Å, 78Å, 79Å, 80Å, 81Å, ​​82Å, 83Å, 84Å, 85Å, 86Å, 87Å, 88Å, 89Å, 90Å, 91Å, 92Å, 93Å, 94Å, 95Å, 96Å, 97Å, 98Å, 99Å, 100Å, 200Å, 300Å, 400Å, 500Å, 600Å, 700Å, 800Å, 900Å, 1000Å, or any number or range between any two of these values. In some embodiments, the polymerase and nucleotide are covalently bonded, and the distance between the linked nucleotide atom and the polymerase is approximately 4 Å to approximately 100 Å. In some embodiments, the distance between the linked nucleotide atom and the polymerase is approximately 5 Å to approximately 20 Å. In some embodiments, the distance between the linked nucleotide atom and the polymerase is approximately 20 Å to approximately 50 Å. In some embodiments, the distance between the linked nucleotide atom and the polymerase is approximately 50 Å to approximately 75 Å. In some embodiments, the distance between the linked nucleotide atom and the polymerase is approximately 75 Å to approximately 100 Å.

[0342] In some embodiments, the linker length is defined as its persistence length. The persistence length corresponds to the root mean square (RMS) distance between the ends of the linker, characterized by dynamic simulation, 2D trapping experiments, or abu initio calculations, where abu initio calculations are based on the statistical distribution of the polymer in a compressible, collapsed, or fluid state required by the conditions of the present solution, suspension, or fluid. In some embodiments, the persistence length of the linker can be at least 0.1 nm, at least 0.2 nm, at least 0.4 nm, at least 1 nm, at least 2 nm, at least 4 nm, at least 10 nm, at least 20 nm, at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 200 nm, at least 300 nm, at least 400 nm, at least 500 nm, at least 700 nm, or at least 1,000 nm, or within a range defined by any two or more of these values, or within a range including any two or more of these values. In some embodiments, the linker for linking nucleotides to the enzyme can have a sustained length of approximately 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. In some embodiments, the linker may have a duration shorter than approximately 5 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 80 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 700 nm, or 1,000 nm. In some embodiments, the linker attached to one nucleotide may be longer or shorter than the linker attached to another nucleotide.In some embodiments, the linker attached to one polymerase may be longer or shorter than the linker attached to another polymerase.

[0343] In some embodiments, the complex is Represented by TIFF2026522421000043.tif42165.

[0344] In some embodiments, the complex is represented by the structure of formula (I) or (II) below, TIFF2026522421000044.tif42165In formula, L 1 C may be substituted. 1-6 Alkylene chain, may be substituted C 2-6 Alkenylene chain and optionally substituted C 1-6 Selected from a group consisting of alkynylene chains, where 1 to 4 methylene units are independently -O-, -N(R a It may be replaced by -C(O)-, -S-, -S(O)-, -S(O)2-, or phenylene. L 2 It is a linker that can be cut, L 3 pol to L 2 It is a linker that connects to, Each R a These are, independently, hydrogen or C 1-6 It is alkyl, R 2 is hydrogen or methyl, R is ribose polyphosphate or deoxyribose polyphosphate. Pol is a polymerase.

[0345] In some embodiments, the complex is The selection is made from the group consisting of TIFF2026522421000045.tif209165 and TIFF2026522421000046.tif168165.

[0346] Prevention of secondary structure formation and removal of residual groups One problem that can occur in enzymatic polynucleotide de novo synthesis is that the synthesized polynucleotides begin to form secondary structures, inhibiting the elongation reaction of template-independent polymerases, which have low activity on the double-strand structure. Increasing the temperature has been studied to reduce secondary structures (Barthel et al., Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3'Terminal Structures for Enzymatic De Novo DNA Synthesis, Genes 2020, 11(1), 102). However, high temperatures induce DNA damage (and quickly damage RNA), and secondary structures remain even at temperatures suitable for synthesis. Thermally stable polymerases are also required for high-temperature synthesis reactions. Wild-type template-independent polymerases, such as terminal deoxynucleotidyl transferase (TdT), are not thermally stable. The use of bases with extracyclic amines masked as azide groups has also been investigated (Nuclera Nucleics PCT Publication WO2020229831A1). However, the demasking reagent (TCEP) causes DNA damage, raising doubts about the stability of azide modifications.

[0347] In some embodiments, this specification provides an improved method for the enzymatic synthesis of long polynucleotides, which involves repeated stepwise elongation using a template-independent polymerase and modified nucleotides to prevent the formation of secondary structures. As shown herein, the use of modified nucleotides having protecting groups on one or more oxygen or nitrogen atoms of the nucleic acid bases suppresses the formation of secondary structures in the nascent chain, which can inhibit the elongation reaction. Such protecting groups prevent the formation of secondary structures such as Watson-Crick base pairing or G-quadrilateral formation. After synthesis is complete, the nucleic acid bases can be returned to their original form by removing the protecting groups, facilitating further use and / or downstream processing of the synthesized polynucleotide.

[0348] In some embodiments, this specification provides modified base-pair protecting groups on nucleic acid bases to inhibit secondary structure during polynucleotide synthesis. These can be efficiently removed after synthesis, leaving polynucleotides free of modified nucleotides that could interfere with downstream use. This specification demonstrates that such modified nucleotides are useful in both enzymatic polynucleotide synthesis using free nucleotides and polynucleotide synthesis using complexes.

[0349] In some embodiments, this specification provides compositions and methods for oligonucleotide synthesis that suppress secondary structure formation and improve the length and accuracy of oligonucleotide synthesis by conferring protecting groups (suppressing secondary structure formation) to the nucleic acid bases of the synthesized polynucleotide. In some embodiments, they are provided as modified nucleotides incorporated into oligonucleotides during enzymatic synthesis. In some embodiments, they are provided as part of a linker-nucleotide complex used during enzymatic oligonucleotide synthesis. Thereafter, the linker is bonded to a base-pairing nitrogen or oxygen atom on the nucleic acid base, and when the linker is cleaved during synthesis to separate the polymerase from the nucleotide, the portion of the linker bonded to the base-pairing nitrogen or oxygen atom remains, which can act as a protecting group (also referred to herein as a “residual group”) that suppresses secondary structure formation, as shown in Figure 2.

[0350] In some embodiments, the disclosure includes a method for synthesizing a polynucleotide, which comprises obtaining a polynucleotide comprising one or more protected nucleic acid bases, and removing one or more protecting groups from the protected nucleic acid bases. In some embodiments, obtaining a polynucleotide comprises contacting a precursor polynucleotide with a nucleotide comprising the protected nucleic acid bases and a template-independent polymerase, and adding a nucleotide to the 3' end of the precursor polynucleotide by the template-independent polymerase. In some embodiments, the method for synthesizing a polynucleotide further comprises repeating the contact step and the addition step one or more times.

[0351] In this specification, the term “protected” nucleotide refers to a nucleotide having a biomolecule bonded to a base-pairing oxygen or nitrogen on a nucleic acid base. In some embodiments, the biomolecule inhibits hydrogen bonding of oxygen or nitrogen to other nucleotides (e.g., Watson-Crick base pairing, G-quadrilateral formation, and other types of hydrogen bonding that can generate secondary structures). Thus, in some embodiments, the biomolecule inhibits the formation of secondary structures during oligonucleotide synthesis. Both “residual group” nucleotides and “protected” nucleotides can refer to the same structure if the linker is bonded to an oxygen or nitrogen on a nucleic acid base. Thus, cleavage of the linker leaves behind a “residual group” nucleotide, which is also a “protected” nucleotide. In some embodiments, the linker of the complex is bonded to a base-pairing oxygen or nitrogen, and the presence of the residual group results in a protected nucleotide that inhibits secondary structure formation during synthesis. Alternatively, a protected nucleotide can refer to a modified nucleotide used during oligonucleotide synthesis, which is not part of the complex but is useful for preventing secondary structure formation during oligonucleotide synthesis.

[0352] In some embodiments, the disclosure includes a method for synthesizing a polynucleotide, the method comprising contacting a precursor polynucleotide with a nucleotide and a polymerase, wherein the nucleotide has a protecting group bonded to a base-pairing oxygen or nitrogen on a nucleic acid base. In some embodiments, the method for synthesizing a polynucleotide comprises adding a nucleotide to the precursor polynucleotide and then removing the protecting group (e.g., a bonded polymerase or a reversible terminator). In some embodiments, the method for synthesizing a polynucleotide comprises repeating the contact, addition, and optionally removal of the protecting group as described herein one or more times. In some embodiments, the removal of one or more protecting groups as described herein includes exposing the polynucleotide to chemical or photodegradation conditions that can remove one or more protecting groups from a protected nucleic acid base.

[0353] While synthesis can be completed entirely using protected nucleotides, synthesis using a combination of unmodified and protected nucleotides can also be effectively used to improve polynucleotide synthesis. In some embodiments, only one of the four nucleotides to be added (e.g., from G or T) is protected during synthesis. In some embodiments, protected nucleotides are added only to target sites where secondary or tertiary structures that may interfere with synthesis are predicted. Such structures can be predicted in various ways based on the presence of complementary DNA regions, and tools exist for each, e.g., the NUPACK algorithm (http: / / www.nuack.org / home / model). Therefore, in some embodiments, only one or two protected nucleotides can be used for the synthesis of complete polynucleotides with improved synthesis. In some embodiments, about 5%, 10%, 20%, 30%, 50%, substantially all, or 100% of a particular nucleotide are incorporated into the polynucleotide in their protected form. In some embodiments, less than 5%, less than 10%, less than 20%, less than 30%, or less than 50% of a particular nucleotide is incorporated into the polynucleotide in its protected form. In some embodiments, more than 5%, more than 10%, more than 20%, more than 30%, or more than 50% of a particular nucleotide is incorporated into the polynucleotide in its protected form. In some embodiments, only protected guanine nucleotides are used in the nucleotide synthesis reaction. Removal of protecting groups at the terminal positions of nucleic acids may be more difficult than removal from internal positions of DNA. Therefore, in some embodiments, nucleotide synthesis is carried out such that the last and first positions 1, 2, or 3 of the synthesized nucleic acid do not contain protected nucleotides.

[0354] In some embodiments, the disclosure includes a method for synthesizing a polynucleotide, which comprises contacting a precursor polynucleotide with a complex comprising a nucleotide covalently bonded to a polymerase via a cleavable linker, thereby cleaving the nucleotide from the polymerase to produce a nucleic acid base having residual groups. In some embodiments, the method for synthesizing a polynucleotide comprises adding a nucleotide to the precursor polynucleotide, followed by cleaving the cleavable linker. In some embodiments, the method for synthesizing a polynucleotide comprises repeating the contact step, addition step, and optionally cleavage step described herein one or more times. In some embodiments, the removal of one or more residual groups described herein comprises contacting the polynucleotide with chemical or photodegradation conditions that can remove one or more residual groups from a nucleic acid base having residual groups.

[0355] In some embodiments, the synthesis of polynucleotides involves stepwise adding nucleotides to a starting molecule (e.g., the initial oligonucleotide) by repeating the steps of conferring a polymerase-nucleotide complex to an oligonucleotide, catalyzing the nucleotide with polymerase to attach the nucleotide to the 3' end of the oligonucleotide, and cleaving the polymerase from the added nucleotide. These steps can be repeated until the desired polynucleotide is synthesized. As described herein, using nucleotides containing protected nucleic acid bases during polynucleotide synthesis helps improve the efficiency and precision of the synthesis by suppressing the formation of secondary structures that may interfere with the addition of input nucleotides by polymerase during synthesis.

[0356] If polymerase is cleaved from a nucleotide during synthesis by the complex, a portion of the linker may remain bound to the nucleotide, leaving a residual group relative to the native nucleotide. This may adversely affect the downstream use of the synthesized polynucleotide (e.g., amplification of the synthesized polynucleotide) or its direct use for its intended purpose. Therefore, improvements to the complex and method are needed for polymerase synthesis by the complex, and such improvements should include the removal of residual groups during or after synthesis.

[0357] One advantage of the methods and compositions provided herein is that they enable the synthesis of polynucleotides without residual groups when polymerase-nucleotide complexes are used in synthesis. This specification provides a preferred linker structure and a method for removing residual / protecting groups remaining after oligonucleotide synthesis, leaving behind native polynucleotides without residual groups.

[0358] In this specification, the term “having a residual group” nucleotide refers to a nucleotide having a portion of the linker that remains bound to the nucleotide after the linker has been cleaved and the bound biomolecule (e.g., polymerase of the polymerase-nucleotide complex) has been released.

[0359] Therefore, in some embodiments, the method for synthesizing polynucleotides using one or more complexes provided herein involves cleaving the nucleotides with polymerase, leaving nucleotides with residual groups in the polynucleotide. The resulting polynucleotide can then be treated under preferred conditions as described herein to remove the residual groups from the modified nucleotides, thereby obtaining polynucleotides having unmodified nucleic acid bases. In some embodiments, removing one or more residual groups from the synthesized polynucleotide involves contacting the polynucleotide with suitable conditions that can remove one or more residual groups from nucleic acid bases having residual groups.

[0360] In some embodiments, this specification provides a method for synthesizing a polynucleotide comprising: obtaining a polynucleotide comprising one or more nucleic acid bases having residual groups; and removing one or more residual groups from nucleic acid bases having residual groups. In some embodiments, obtaining a polynucleotide comprises: contacting a precursor polynucleotide with a nucleotide comprising nucleic acid bases bound to a template-independent polymerase; and adding a nucleotide to the 3' end of the precursor polynucleotide by the template-independent polymerase. In some embodiments, the method for synthesizing a polynucleotide further comprises repeating the contact step and the addition step one or more times.

[0361] Enzyme-removable protecting groups In some embodiments, this specification provides modified nucleotides or nucleotide linkers in which, upon cleavage of the linker, a protecting group or residual group remains on the nucleic acid base that can be removed by an enzyme.

[0362] Furthermore, this specification provides an improved method for nucleic acid synthesis by repeated extension using template-independent polymerase. As shown herein, the use of modified nucleotides in which the extracyclic oxygen of the nucleic acid base is methylated or alkylated (other than methylated) suppresses the formation of secondary structures in the nascent chain that can inhibit the extension reaction. Such modified nucleotides prevent Watson-Crick base pairing and / or other structures (e.g., G-quadrilateral formation). After synthesis is complete, the nucleic acid base can be returned to its original form by enzymatically removing the alkyl group.

[0363] This disclosure encompasses methods for synthesizing polynucleotides containing one or more alkylated nucleic acid bases and for removing one or more alkyl groups from the alkylated nucleic acid bases. In some embodiments, the inclusion of one or more alkylated nucleic acid bases prevents base pairing or the formation of undesirable secondary structures during synthesis. In some embodiments, the alkylated nucleic acid bases belong to classes and subclasses as described below herein. In some embodiments, obtaining a polynucleotide involves contacting a precursor polynucleotide with a nucleotide containing an alkylated nucleic acid base and a template-independent polymerase, and adding a nucleotide to the 3' end of the precursor polynucleotide by the template-independent polymerase. In some embodiments, the method for synthesizing a polynucleotide further includes repeating the contact step and the addition step one or more times.

[0364] After completing the synthesis of a polynucleotide using one or more alkylated nucleotides, the resulting polynucleotide can be treated with an alkyltransferase to remove the alkyl group attached to the nucleotide, thereby obtaining a polynucleotide having an unmodified nucleic acid base. In some embodiments, removing one or more alkyl groups from the synthesized polynucleotide involves contacting the polynucleotide with one or more enzymes capable of removing one or more alkyl groups from alkylated nucleic acid bases. In some embodiments, the enzyme suitable for dealkylation of alkylated nucleic acid bases is an alkyltransferase. In some embodiments, the enzyme suitable for dealkylation of polynucleotides is an alkyltransferase belonging to EC 2.1.1.63. In some embodiments, the alkyltransferase is an AGT (alkylguanine transferase), for example, O 6 - AlkB is an alkylguanine DNA alkyltransferase. In some embodiments, the enzyme used to remove alkyl groups from alkylated nucleic acid bases is AlkB (Escherichia coli), which is an alpha-ketoglutarate-dependent hydroxylase that oxidatively dealkylates the DNA substrate.

[0365] The reactions shown in Figures 3 and 4 involve the cleavage of a linker in the nucleotide-TdT complex and the removal of the alkyl group from the alkylated nucleotide by an AGT enzyme. In Figure 3, the linker is distinct from the alkyl modifying group of the nucleotide. In Figure 4, TdT is linked to the alkyl modifying group of the nucleotide, and the alkyl group remains after the linker bound to the nucleotide and TdT is cleaved.

[0366] Enzymes suitable for dealkylation of alkylated nucleic acid bases after synthesis can be determined by screening a set of enzymes known to be involved in the dealkylation reaction of nucleic acid bases or reactions closely related thereto. An example of a simple screening method is described in Example 8 of this specification. Using such a screening method, those skilled in the art can identify enzymes suitable for dealkylation and for carrying out the DNA synthesis method.

[0367] For example, O 6 -While alkylguanine DNA alkyltransferase and AlkB are exemplary enzymes suitable for dealkylation, several other enzymes from various species are quite similar and may be suitable for dealkylation of synthetic nucleotides. Tables 1 and 2 below list alkyltransferases that may be suitable for dealkylation of polynucleotide synthetic products described herein.

[0368] [Table 1] TIFF2026522421000048.tif206157TIFF2026522421000049.tif83157

[0369] [Table 2] TIFF2026522421000051.tif210157

[0370] In some embodiments, the alkylated nucleic acid base is of the following formula (V) or (VI): Represented by TIFF2026522421000052.tif42165, in the formula, X is -C(R 2 ) = or -N = R 1 C 1-6 Alkyl, C 2-6 Alkenil, C 1-6 Alkinyl and -(CH2) 0-3 Selected from the group consisting of Ph, where R 1 This is 1 to 6 R 1a It may also be replaced with Each R 1a These are, independently, halogen, C 1-6 Alkyl, -(CH2) 0-3 Ure 1b -NO2, -N3, -OPO2OH, and -(CH2) 0-3 NHR 1b Selected from, Each R 1b These are, independently, hydrogen and C 1-6 Alkyl, -C(O)(C 1-6 Alkyl), C 1-6 Haloalkyl, -C(O)(C 1-6 Selected from haloalkyl and -CH2OAc, R 2 C is hydrogen, which may be substituted. 1-4 Selected from alkyl chains, where 1-2 methylene units are independently -O-, -N(R) a )-, -C(O)-, -S-, -S(O)-, -S(O)2-, or phenylene may be substituted, R 2 This is 1 to 6 R 2a It may also be replaced with Each R 2a These are, independently, halogen, C 1-6 Alkyl, -(CH2) 0-3 Ure 2b -NO2, -N3, -OPO2OH, and -(CH2) 0-3 NHR 2b Selected from, Each R 2b These are, independently, hydrogen and C 1-6 Selected from alkyl groups.

[0371] In some embodiments, R 1 C 1-4 It is alkyl. In some embodiments, R 1 R is selected from the group consisting of methyl, ethyl, n-propyl, and n-butyl. In some embodiments, R 1 is methyl. In some embodiments, R 1 is ethyl. In some embodiments, R 1 is n-propyl. In some embodiments, R 1 It is n-butyl.

[0372] In some embodiments, R 1 teeth, Selected from the group consisting of TIFF2026522421000053.tif58165.

[0373] In some embodiments, R 1 teeth, Selected from the group consisting of TIFF2026522421000054.tif53165.

[0374] In some embodiments, X is -C(R 2 )= or -N=. In some embodiments, X is -C(R 2 ) In some embodiments, X is -N=.

[0375] In some embodiments, R 2 This is selected from the group consisting of C1-C2 alkyl groups which may be substituted with hydrogen or -OH.

[0376] In some embodiments, the alkylated nucleic acid base is The selection is made from the group consisting of TIFF2026522421000055.tif216165 and TIFF2026522421000056.tif111165.

[0377] In some embodiments, the linker is bonded to a different base position than the alkylation site.

[0378] In some embodiments, the linker is specifically bound to an amino acid of the polymerase. In such cases, it is preferable to bind the linker to an amino acid at a position that can be mutated without loss of polymerase activity (e.g., positions 180, 188, 253, or 302 of mouse TdT (same numbering as in crystal structure PDB ID: 4127)). To avoid interfering with catalytic activity, it is preferable not to bind the linker to an amino acid involved in the catalytic activity of the polymerase. Residues known to be involved in catalytic activity, and methods for determining whether a residue is involved in catalytic activity (e.g., by site-directed mutagenesis), are obvious to those skilled in the art and are outlined in the 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)).

[0379] In some embodiments, the linker of the complex can bind to position 7 of deaza dGTP, or to position 5 of dTTP or dUTP.

[0380] In some embodiments, the linker of the complex leaves behind residual groups when cleaved. In such cases, residual groups may remain on the DNA after cleavage. In some embodiments, the residual groups include hydroxyl groups. In some embodiments, the residual groups include amine groups. In some embodiments, the residual groups include hydroxyalkyl groups.

[0381] In some embodiments, the linker of the complex is bound to the alkyl group of the O-alkylated nucleic acid base. In certain embodiments, the linker of the complex bound to the O-alkylated nucleic acid base leaves a residual group when cleaved. In some embodiments, the residual group is removed using an enzyme capable of removing one or more alkyl groups from the alkylated nucleic acid base.

[0382] In some embodiments, the complex has the structure of formula (I) or (II) below: Represented by TIFF2026522421000057.tif53165, in the formula, L 1 C may be substituted. 1-6 Alkylene chain, may be substituted C 2-6 Alkenylene chain and optionally substituted C 1-6 Selected from a group consisting of alkynylene chains, where 1 to 4 methylene units are independently -O-, -N(R a It may be replaced by -C(O)-, -S-, -S(O)-, -S(O)2-, or phenylene. L 2 It is a linker that can be cut, X is either -N= or -C(H)=, Each R a These are, independently, hydrogen or C 1-6 It is alkyl, R 2 is hydrogen or methyl, R is ribose polyphosphate or deoxyribose polyphosphate. Pol is a polymerase.

[0383] In some embodiments, the complex is Represented by TIFF2026522421000058.tif53165.

[0384] In some embodiments, L 1 teeth, Selected from the group consisting of TIFF2026522421000059.tif58165.

[0385] In some embodiments, the complex is The selection is made from the group consisting of TIFF2026522421000060.tif218165 and TIFF2026522421000061.tif225165.

[0386] In some methods, ribose polyphosphate is selected from the group consisting of ribose triphosphate, ribose tetraphosphate, ribose pentaphosphate, and ribose hexaphosphate. In some methods, ribose polyphosphate is ribose triphosphate. In some methods, ribose polyphosphate is ribose hexaphosphate. In some methods, ribose polyphosphate is ribose pentaphosphate. In some methods, ribose polyphosphate is ribose tetraphosphate.

[0387] In some embodiments, the Disclosure includes a method for processing polynucleotides synthesized using alkylated nucleic acid bases, the method being To produce a polynucleotide containing one or more alkylated nucleic acid bases, This includes removing one or more alkyl groups from one or more alkylated nucleic acid bases.

[0388] In some embodiments, the disclosure includes a method for synthesizing polynucleotides comprising alkylated nucleic acid bases, the method being The process involves contacting a precursor polynucleotide with polymerase and a nucleotide containing an alkylated nucleic acid base, This includes adding a nucleotide to the 3' end of a precursor polynucleotide using polymerase.

[0389] In some embodiments, the disclosure includes a method for synthesizing a polynucleotide, comprising contacting a precursor polynucleotide with a complex comprising a nucleotide covalently bonded to a polymerase via a cleavable linker, wherein the nucleotide comprises an alkylated nucleic acid base. In some embodiments, the method for synthesizing a polynucleotide comprises adding a nucleotide to the precursor polynucleotide, followed by cleaving the cleavable linker. In some embodiments, the method for synthesizing a polynucleotide comprises repeating the contact step, addition step, and optionally cleavage step described herein one or more times. In some embodiments, the removal of one or more alkyl groups as described herein comprises contacting the polynucleotide with an enzyme capable of removing one or more alkyl groups from an alkylated nucleic acid base. In some embodiments, the method for synthesizing a polynucleotide comprises contacting the polynucleotide with two or more enzymes capable of removing one or more alkyl groups from an alkylated nucleic acid base.

[0390] In some embodiments, the synthesis of polynucleotides involves stepwise adding nucleotides to a starting molecule (e.g., the initial oligonucleotide) by repeating the steps of conferring a polymerase-nucleotide complex to an oligonucleotide, catalyzing the nucleotide with polymerase to attach the nucleotide to the 3' end of the oligonucleotide, and cleaving the polymerase from the added nucleotide. These steps can be repeated until the desired polynucleotide is synthesized. As described herein, using nucleotides containing alkylated nucleic acid bases during polynucleotide synthesis helps improve the efficiency and precision of the synthesis by suppressing the formation of secondary structures that may interfere with the addition of input nucleotides by polymerase during synthesis.

[0391] While synthesis can be completed entirely using alkylated nucleotides, synthesis using combinations of unmodified and alkylated nucleotides can also be effectively used to improve polynucleotide synthesis. In some embodiments, only one of the four nucleotides to be added (e.g., from G or T) is alkylated during synthesis. In some embodiments, alkylated nucleotides are added only to target sites where secondary or tertiary structures that may interfere with synthesis are predicted. Such structures can be predicted in various ways based on the presence of complementary DNA regions, and tools exist for each, e.g., the NUPACK algorithm (http: / / www.nuack.org / home / model). Thus, in some embodiments, only one or two alkylated nucleotides can be used for the synthesis of complete polynucleotides with improved synthesis. In some embodiments, about 5%, 10%, 20%, 30%, 50%, substantially all, or 100% of a particular nucleotide are incorporated into the polynucleotide in its alkylated form. In some embodiments, less than 5%, less than 10%, less than 20%, less than 30%, or less than 50% of a particular nucleotide is incorporated into the polynucleotide in its alkylated form. In some embodiments, more than 5%, more than 10%, more than 20%, more than 30%, or more than 50% of a particular nucleotide is incorporated into the polynucleotide in its alkylated form. In some embodiments, only alkylated guanine nucleotides are used in the nucleotide synthesis reaction. Removing alkyl groups at the terminal positions of nucleic acids by alkyltransferase may be more difficult than removing them from internal positions of DNA. Therefore, in some embodiments, nucleotide synthesis is carried out such that the last and first positions 1, 2, or 3 of the synthesized nucleic acid do not contain alkylated nucleotides.

[0392] In some embodiments, the nucleotide analogs described herein have a reversible terminator group (e.g., an O-azidomethyl group or an O-NH2 group) at the 3' position of the sugar, or a (alpha-tert-butyl-2-nitrobenzyl)oxymethyl group at the 5 position of the pyrimidine or the 7 position of the 7-deazaprine (see, for example, Chen et al., Genomics, Proteomics & Bioinformatics 2013 11:34-40 for an overview). In such embodiments, the nucleotide analogs, once incorporated into the nucleic acid, inhibit or interfere with further elongation and control the termination of synthesis. In some embodiments, the RTdNTP-polymerase complex used as part of the complex brings about termination independently of shielding effects, for example, when the 3'-modified RTdNTP is tethered to the polymerase, and the length of the linker used may exceed 100 Å or 200 Å.

[0393] O 6 Alkylguanine-DNA alkyltransferases (AGTs) irreversibly transfer alkyl groups from their substrate modified nucleotides. This specification describes alkyl-modified nucleotides useful in synthesis, where the alkyl group can be removed by AGT. In some embodiments, this removal converts the "residual group" nucleotide to its native form or nucleic acid base in the synthetic oligonucleotide. Several forms of enzymes can be used (e.g., human, mouse, rat, chimeric, or other species AGTs) as long as they have similar properties in their reaction with the substrate alkyl group. In this specification, alkyl group refers to any group that can act as a substrate and is removed from a nucleotide by an AGT enzyme.

[0394] Furthermore, in this disclosure, O6-alkylguanine-DNA alkyltransferase includes variants of wild-type AGT. Such variants may differ by substitution, deletion, or addition of one or more amino acids, but still possess the property of transferring labels present in the substrate to the AGT portion of the fusion protein. AGT variants can be obtained by chemical modification using techniques well known to those skilled in the art. Preferably, AGT variants can be prepared using protein engineering techniques known to those skilled in the art and / or by utilizing molecular evolution for generating and selecting novel O6-alkylguanine-DNA alkyltransferases. Such techniques include, for example, saturated mutagenesis, error-prone PCR for introducing mutations at arbitrary locations in the sequence, DNA shuffling used after saturated mutagenesis and / or error-prone PCR, or family shuffling using genes from several species.

[0395] In some embodiments, the alkylated nucleic acid base is a nucleic acid base of formula (I) or formula (II), TIFF2026522421000062.tif47165In formula, R 1 C 1-6 Alkyl, C 2-6 Alkenil, C 1-6 Alkinyl and -(CH2) 0-3 Selected from the group consisting of Ph, where R 1 This is 1 to 6 R 1a It may also be replaced with Each R 1a These are, independently, halogen, C 1-6 Alkyl, -(CH2) 0-3 Ure 1b -NO2, -N3, -OPO2OH, and -(CH2) 0-3 NHR 1b Selected from, Each R 1b These are, independently, hydrogen and C 1-6 Alkyl, -C(O)(C 1-6 Alkyl), C 1-6 Haloalkyl, -C(O)(C 1-6Selected from haloalkyl and -CH2OAc, R 2 is hydrogen or methyl, X is either -N= or -C(H)=, R is either ribose polyphosphate or deoxyribose polyphosphate.

[0396] In some embodiments, X is -C(H)= or -N=. In some embodiments, X is -C(H)=. In some embodiments, X is -N=.

[0397] In some embodiments, R 1 C 1-6 Alkyl, C 2-6 Alkenil, C 1-6 Alkinyl and -(CH2) 0-3 Selected from the group consisting of Ph, where R 1 This is 1 to 6 R 1a It may be replaced with R. In some embodiments, 1 C 2-6 Alkyl, C 2-6 Alkenil, C 2-6 Alkinyl and -(CH2) 0-3 Selected from the group consisting of Ph, where R 1 This is 1 to 6 R a It may be replaced with R. In some embodiments, 1 C 1-3 Alkyl, C 2-4 Alkenil, C 2-4 Selected from the group consisting of alkynyl and -(CH2)Ph, where R 1 This is one R a It may be replaced with R. In some embodiments, 1 R is selected from the group consisting of methyl, ethyl, C4 alkenyl, C4 alkynyl, and -CH2Ph, where R 1 This is one R a It may be replaced with R. In some embodiments, 1 R is selected from the group consisting of ethyl, C4 alkenyl, C4 alkynyl, and -CH2Ph, where R1 This is one R a It may be replaced with R. In some embodiments, 1 R is selected from the group consisting of C1-C3 alkyl groups, where R 1 This is 1 to 3 R a It may be replaced with R. In some embodiments, 1 R is selected from the group consisting of C1-C3 alkyl groups, where R 1 This is 1 to 3 R a It may be replaced with .

[0398] In some embodiments, R 1 teeth, Selected from the group consisting of TIFF2026522421000063.tif37165.

[0399] In some embodiments, R 1 This is 1 to 3 R a Selected from the group consisting of methyl, ethyl, and n-propyl, which may be substituted with methyl.

[0400] In some embodiments, R 1 It is methyl.

[0401] In some embodiments, the nucleotide is The selection is made from the group consisting of TIFF2026522421000064.tif220165.

[0402] Chemically removable protecting groups In some embodiments, preferred conditions for removing a protecting group from a protected nucleic acid base include exposing the protected nucleic acid base to light. In some embodiments, the light is ultraviolet light. In some embodiments, the ultraviolet light has a wavelength of about 365 nm.

[0403] In some embodiments, preferred conditions for removing a protecting group from a protected nucleic acid base include treating the protected nucleic acid base with an acidic oxidizing solution. In some embodiments, preferred conditions for removing a protecting group from a protected nucleic acid base include treating the protected nucleic acid base with a nitrite solution.

[0404] In some embodiments, preferred conditions for removing a protecting group from a protected nucleic acid base include treating the protected nucleic acid base with a reducing agent. In some embodiments, the reducing agent is a phosphorus-based reducing agent. In some embodiments, the phosphorus-based reducing agent is PPh3 or TCEP. In some embodiments, the phosphorus-based reducing agent is TCEP.

[0405] In some embodiments, preferred conditions for removing the protecting group include a step of treating the protected nucleic acid base with an oxidizing agent. In some embodiments, the oxidizing agent can oxidize a thioether (i.e., sulfide) to a sulfone. In some embodiments, the oxidizing agent is selected from the group consisting of H2O2, mCPBA, and TAPC. In some embodiments, the conditions for removing the protecting group further include an alkaline (or high pH) environment. In some embodiments, the alkaline environment includes a pH of about 8. In some embodiments, the alkaline environment is an environment with a pH higher than the pH during polynucleotide synthesis.

[0406] In some embodiments, preferred conditions for removing the protecting group include a step of treating the protected nucleic acid base with a base. In some embodiments, the base is selected from the group consisting of NH4OH, KOH, NaOH, KOMe, NaOMe, and KOtBu.

[0407] In some embodiments, preferred conditions for removing the protecting group include a step of treating the protected nucleic acid base under conditions that remove the allyl group. In some embodiments, preferred conditions for removing the protecting group include a step of treating the protected nucleic acid base with Pd. In some embodiments, preferred conditions for removing the protecting group include a step of treating the protected nucleic acid base with Pd(OAc), Pd2(dba)3, and Pd2(pmdba)3.

[0408] This specification describes structures of removable protecting groups or residual groups that can be chemically removed from the nitrogen or oxygen of the nucleic acid base, leaving the native nucleotide. For polymerase-nucleotide complexes, the linker that links the nucleotide to the polymerase is attached to the nucleotide at the nitrogen or oxygen of the nucleic acid base, and therefore, cleavage of the linker leaves a removable protecting group on the nitrogen or oxygen. In some embodiments, the protecting group / residual group structures described herein remain attached to the nitrogen or oxygen of the nucleic acid base during synthesis but are removed before downstream processing or use of the newly synthesized oligonucleotide. In some embodiments, the nucleotide has a removable protecting group structure described herein attached to the nitrogen or oxygen atom of the nucleic acid base (having a structure corresponding to the removable residual group after cleavage of the linker of the complex), and such nucleotides can be used in oligonucleotide synthesis.

[0409] In some embodiments, the polymerase-nucleotide complex has the following structure: As represented in TIFF2026522421000065.tif37165, it has a linker bonded to a nucleic acid base, where Y is a nucleic acid base, L is a linker bonded to a base-pairing nitrogen or oxygen of the nucleic acid base, R is ribose polyphosphate or deoxyribose polyphosphate, and Pol is polymerase.

[0410] Linker L is given by formula ZL 1 -L 2 -L3 It can be expressed as, in the formula, ZL 1 -L 2 L represents the portion of the linker remaining after the linker has been cleaved and polymerase has been separated from the nucleotides. 3 L 2 It is a linker that can be cleaved, and it is bound to polymerase.

[0411] Nucleotides with residual groups and protected nucleotides are also described herein. Nucleotides with residual groups are nucleotides that have a residue of the linker (i.e., residual groups after the linker has been cleaved and the polymerase has been separated from the nucleotide). Protected nucleotides are nucleotides that have a protecting group bonded to the base-pairing oxygen or nitrogen of the nucleic acid base. Protected nucleotides have a protecting group structure, which corresponds to the structure of nucleotides with residual groups.

[0412] In some embodiments, the nucleotide having residual groups or the protected nucleotide has the following structure: It has nucleic acid bases represented by TIFF2026522421000066.tif32165, where Y is a nucleic acid base and LR 1 L is a protecting group or residual group, L is the base-pairing nitrogen or oxygen-bonded portion of the nucleic acid base, and R 1 is hydrogen, -OH, -N(R b )2, and -SH are selected from the group, where each R b C is independently hydrogen or may be substituted. 1-6 It is alkyl. L of a protected nucleotide / nucleotide with a residual group is of formula ZL. 1 -L 2 This can be represented by the same ZL in the complex structure. 1 -L 2 Corresponds to ZL 1 -L 2 -R 1Exemplary removable residual / protecting groups, including those mentioned herein, are described herein, as are conditions for their removal from nucleotides. In some embodiments, removal of a photocleavable residual or protecting group is achieved by exposure to light of a corresponding wavelength. In some embodiments, removal is achieved by a suitable set of chemical conditions, such as a basic environment.

[0413] In some embodiments, Z is a bond, -C(O)-, -C(O)CH2-, -C(O)C(R L )2-,-C(O)CH(R L Selected from the group consisting of -, -C(O)O-, and -C(O)N(H)-, L 1 is to combine, Selected from the group consisting of TIFF2026522421000067.tif47165, In the formula, L 1 This is 1 to 4 R L It may also be replaced with each R L These are independently selected from the group consisting of halogens, hydroxyls, oxos, and optionally substituted C1-C3 alkyls, where two R 1 It may also form a 3-6 membered carbocykyl ring together with intervening atoms (multiple atoms are possible), L 2 C may be bonded or substituted. 1-12 Alkylene chain, C4-C 20 Polyethylene glycol, may be substituted C 2-12 Alkenylene chain and optionally substituted C 2-12 Selected from a group consisting of alkynylene chains, where 1 to 6 methylene units are independently -O-, -N(R b )-, -C(O)-, -S-, -S(O)-, -S(O)2-, or phenylene may be substituted, and W may be -O-, -S-, -S(O)2-, and -N(R b Selected from the group consisting of )-, each R a is a halogen, -Me, or -OMe, and each R b C is independently hydrogen or may be substituted. 1-6It is an alkyl group, n is 1 or 2, and here L 1 Z and Z cannot be connected at the same time.

[0414] O-bonded residual group / protecting group In some embodiments, the complex, which includes a linker having a removable residual group, is bound to the oxygen on the nucleic acid base of the nucleotide. In some embodiments, the residual group or protecting group is bound to the oxygen on the nucleic acid base of the nucleotide. In some embodiments, the oxygen is base-pairing oxygen on the nucleic acid base.

[0415] In some embodiments, the linker, residual group, or protecting group is bonded to the O4 oxygen of uracil or adenine, or the O6 oxygen of guanine. Examples of chemically removable residual groups or protecting groups are listed below.

[0416] In some embodiments, the sulfonyl group is used as a protecting or residual group that can be removed from the nucleic acid base. The sulfonyl group bonded to O can be removed in a beta-elimination reaction by a suitable base such as NH4OH.

[0417] In some embodiments, the linker of the complex bonded to oxygen or nitrogen on the nucleic acid base contains a sulfonyl group. In some embodiments, the residual group remaining after cleavage of the linker contains a sulfonyl group. In some embodiments, the protected nucleotide contains an O-bonded protecting group containing a sulfonyl group. In some embodiments, the residual group or protecting group containing a sulfonyl group is removed from the nucleotide by exposure to a suitable base. In some embodiments, the preferred base is a strong base. In some embodiments, the preferred base is a hydroxide with a suitable counterion. In some embodiments, the preferred base is selected from the group consisting of NaOH, NH4OH, KOH, KOtBu, NaOMe, and KOMe.

[0418] In some embodiments, L 1 teeth, The filename is TIFF2026522421000068.tif22165.

[0419] In some embodiments, -ZL 1 -L 2 -R 1 teeth, The filename is TIFF2026522421000069.tif22165.

[0420] An exemplary Z1-L1-L2 structure of a complex having a linker containing a sulfonyl group is shown below. Such a Z1-L1-L2 structure can be used for synthesis with nucleotides having residual groups (after linker cleavage) or protected nucleotides. An exemplary complex having a sulfonyl group bonded to oxygen on the nucleic acid base (which is retained as a residual group on the nucleotide after linker cleavage and can be removed) is shown below. TIFF2026522421000070.tif99165

[0421] Further Z-L1-L2 structures in exemplary nucleic acid bases that represent nucleic acid bases with residual groups or protected nucleic acid bases are shown below. TIFF2026522421000071.tif78165

[0422] This disclosure includes a method for preparing polynucleotides, the method comprising treating a nucleic acid base having a residual group containing a sulfonyl group with a suitable base. Although not bound by any particular theory, treatment of a nucleic acid base having a residual group containing a sulfonyl group with a suitable base results in the following beta-elimination: TIFF2026522421000072.tif47165

[0423] In some embodiments, complexes or nucleotides containing sulfonyl groups can be prepared as outlined in Scheme 1. Scheme 1 TIFF2026522421000073.tif186165

[0424] In some embodiments, the thioether group is used as a protecting group or residual group that can be removed from the nucleic acid base. The thioether group can be removed by exposure to a suitable nucleophile.

[0425] In some embodiments, the linker of the complex bonded to oxygen or nitrogen on the nucleic acid base contains a thioether. In some embodiments, the residual group remaining after cleavage of the linker contains a thioether. In some embodiments, the protected nucleotide contains an O-linked protecting group or an N-linked protecting group containing a thioether. In some embodiments, the residual group or protecting group containing a thioether is removed by exposure to a suitable oxidizing agent followed by exposure to a suitable base.

[0426] In some embodiments, L 1 teeth, This is TIFF2026522421000074.tif17165. In some embodiments, L 1 teeth, The filename is TIFF2026522421000075.tif22165.

[0427] In some embodiments, -ZL 1 -L 2 -R 1 teeth, The filename is TIFF2026522421000076.tif17165.

[0428] An exemplary Z1-L1-L2 structure of a complex having a linker containing a nitrobenzyl photocleavable group is shown below. Such a Z1-L1-L2 structure can be used for synthesis with nucleotides having residual groups (after linker cleavage) or protected nucleotides. An exemplary complex having a photocleavable group bonded to the oxygen on the nucleic acid base (which is retained as a residual group on the nucleotide after linker cleavage and can be removed) is shown below. TIFF2026522421000077.tif89165

[0429] Further Z-L1-L2 structures in exemplary nucleic acid bases that represent nucleic acid bases with residual groups or protected nucleic acid bases are shown below. TIFF2026522421000078.tif74165

[0430] In some embodiments, the disclosure includes a method for preparing polynucleotides, the method including removing residual or protecting groups, including thioether groups, attached to nucleic acid bases by oxidation to generate sulfonyl groups and treatment with a suitable base to remove sulfonyl groups.

[0431] For example, the removal of a sulfonyl group bonded to the oxygen of a nucleic acid base can be achieved by exposing the base to an oxidizing agent and then to a base, as follows: TIFF2026522421000079.tif42165

[0432] In some embodiments, complexes or nucleotides containing sulfonyl groups can be prepared as outlined in Scheme 2. Scheme 2 TIFF2026522421000080.tif53165

[0433] In some embodiments, the cyanoethyl group is used as a protecting group or a residual group that can be removed from the nucleic acid base. The cyanoethyl group bonded to O can be removed by a suitable base such as NH4OH.

[0434] In some embodiments, the linker contains a cyanoethyl group. In some embodiments, the residual group contains a cyanoethyl group. In some embodiments, the residual group contains a cyanoethyl group that is removed by exposure to a suitable base. In some embodiments, the preferred base is a strong base. In some embodiments, the preferred base is a hydroxide with a suitable counterion. In some embodiments, the preferred base is selected from the group consisting of NaOH, NH4OH, KOH, KOtBu, NaOMe, and KOMe.

[0435] In some embodiments, L 1 teeth, The filename is TIFF2026522421000081.tif22165.

[0436] In some embodiments, the nucleotide is Selected from the group consisting of TIFF2026522421000082.tif47165.

[0437] An exemplary removable Z1-L1-L2 structure bonded to the oxygen of a protected nucleic acid base having a linker containing a cyanoethyl group is shown below. Such a Z1-L1-L2 structure can be used for synthesis in a complex that leaves a nucleotide with residual groups (after linker cleavage) or in a protected nucleotide. TIFF2026522421000083.tif58165

[0438] In some embodiments, the protected nucleic acid base or the nucleic acid base having a residual group is Selected from the group consisting of TIFF2026522421000084.tif43165.

[0439] This disclosure includes a method for preparing polynucleotides, the method comprising treating a nucleic acid base having a residual group containing a cyanoethyl group with a suitable base. Although not bound by any particular theory, treatment of a nucleic acid base having a residual group containing a cyanoethyl group with a suitable base results in the following beta-elimination: TIFF2026522421000085.tif73165

[0440] In some embodiments, the cyanoethyl group-containing complex or nucleotide can be prepared as outlined in Scheme 3. Scheme 3 TIFF2026522421000086.tif150165

[0441] In some embodiments, the allyl group is used as a protecting or residual group that can be removed from the nucleic acid base. In some embodiments, the linker contains an allyl group. In some embodiments, the residual group contains an allyl group. In some embodiments, the residual group contains an allyl group that is removed by exposure to a suitable transition metal catalyst. In some embodiments, the suitable transition metal catalyst is a palladium catalyst. In some embodiments, the suitable transition metal catalyst is selected from the group consisting of Pd2(dba)3, Pd2(pmdba)3, PdCl2, Pd(TFA)2, and Na2PdCl4.

[0442] In some embodiments, L 1 teeth, The filename is TIFF2026522421000087.tif17165.

[0443] In some embodiments, L 1 -L 2 teeth, The filename is TIFF2026522421000088.tif22165.

[0444] An exemplary removable Z1-L1-L2 structure bonded to the oxygen of a protected nucleic acid base having a linker containing a cyanoethyl group is shown below. Such a Z1-L1-L2 structure can be used for synthesis in a complex that leaves a nucleotide with residual groups (after linker cleavage) or in a protected nucleotide. TIFF2026522421000089.tif32165

[0445] This disclosure includes a method for preparing polynucleotides, the method comprising treating a nucleic acid base having a residual group containing an allyl group with a transition metal catalyst and optionally one or more suitable ligands. In some embodiments, a suitable ligand is P(PhSO3Na)3. Although not bound by any particular theory, catalytic deallylation is brought about by treating a nucleic acid base having a residual group containing an allyl group with a suitable transition metal catalyst. TIFF2026522421000090.tif27165

[0446] In some embodiments, complexes or nucleotides containing allyl groups can be prepared as outlined in Scheme 4. Scheme 4 TIFF2026522421000091.tif150165

[0447] In some embodiments, the azide is used as a protecting or residual group that can be removed from the nucleic acid base. In some embodiments, the linker contains an azide. In some embodiments, the residual group contains an azide. In some embodiments, the residual group contains an azide that is removed by exposure to a suitable reducing agent. In some embodiments, a suitable reducing agent is phosphine. In some embodiments, a suitable reducing agent is TCEP. TIFF2026522421000092.tif32165

[0448] In some embodiments, L 1 teeth, The filename is TIFF2026522421000093.tif22165.

[0449] An exemplary Z1-L1-L2 structure of a complex having an azide-containing linker is shown below. Such a Z1-L1-L2 structure can be used for synthesis with nucleotides having residual groups (after linker cleavage) or protected nucleotides. An exemplary complex having a photocleavable group bonded to oxygen on the nucleic acid base (which is retained as a residual group on the nucleotide after linker cleavage and can be removed) is shown below. TIFF2026522421000094.tif217165

[0450] Further Z-L1-L2 structures in exemplary nucleic acid bases that represent nucleic acid bases with residual groups or protected nucleic acid bases are shown below. TIFF2026522421000095.tif130165

[0451] This disclosure includes a method for preparing polynucleotides, the method comprising treating a nucleic acid base having a residual group containing an azidyl group with a suitable reducing agent. Although not bound by any particular theory, treatment of a nucleic acid base having a residual group containing an azidyl group with a suitable reducing agent results in the removal of the residual group as follows: TIFF2026522421000096.tif42165

[0452] In some embodiments, the oxime is used as a protecting or residual group that can be removed from the nucleic acid base. In some embodiments, the linker contains an oxime. In some embodiments, the residual group contains an oxime. In some embodiments, the residual group contains an oxime that is removed by exposure to a suitable nucleophile and then to a suitable acid. In some embodiments, the suitable nucleophile is H2NOtBu. In some embodiments, the suitable acid is HONO. TIFF2026522421000097.tif42165

[0453] In some embodiments, L 1 teeth, This is TIFF2026522421000098.tif22165. In some embodiments, L 1 teeth, The filename is TIFF2026522421000099.tif22165.

[0454] An exemplary Z1-L1-L2 structure of a complex having a linker containing an oxime is shown below. Such a Z1-L1-L2 structure can also be used for synthesis with nucleotides having residual groups (after linker cleavage) or protected nucleotides. An exemplary complex having an oxime bound to oxygen on the nucleic acid base (which is retained as a residual group on the nucleotide after linker cleavage and can be removed) is shown below. TIFF2026522421000100.tif217165

[0455] Further Z-L1-L2 structures in exemplary nucleic acid bases that represent nucleic acid bases with residual groups or protected nucleic acid bases are shown below. TIFF2026522421000101.tif155165

[0456] This disclosure includes a method for preparing polynucleotides, the method comprising treating a nucleic acid base having a residual group containing an oxymyl group with a suitable nucleophile, followed by treatment with a suitable acid. Although not bound by any particular theory, the removal of the oxymyl group can be achieved as shown below.

[0457] In some embodiments, the silyl group is used as a protecting group or residual group that can be removed from the nucleic acid base. In some embodiments, the linker contains a silyl group. In some embodiments, the residual group contains a trimethylsilyl group. In some embodiments, the linker contains a silyl group. In some embodiments, the residual group contains a trimethylsilyl group. In some embodiments, the residual group contains a silyl group that is removed by exposure to a halogen source. In some embodiments, the residual group contains a silyl group that is removed by exposure to ZnBr2. In some embodiments, the residual group contains a silyl group that is removed by exposure to a suitable fluoride source. In some embodiments, the suitable fluoride source is selected from the group consisting of KF, TBAF, and TBAT. TIFF2026522421000102.tif32165

[0458] Residual group / protecting group bonded to N In some embodiments, the complex, which includes a linker having a removable residual group, is bonded to the nitrogen on the nucleic acid base of the nucleotide. In some embodiments, the residual group or protecting group is bonded to the nitrogen on the nucleic acid base of the nucleotide. In some embodiments, the nitrogen is a base-pairing nitrogen on the nucleic acid base.

[0459] In some embodiments, the linker, residual group, or protecting group is bonded to the N1 or N2 nitrogen of guanine, the N4 nitrogen of cytosine, or the N6 nitrogen of adenine. Examples of chemically removable residual groups or protecting groups are listed below.

[0460] In some embodiments, Z is a carbamate bonded to the nitrogen of a nucleic acid base. When Z is a carbamate, the above-described O-bonded protecting group or residual group structure (-L1 or -L1-L2) can be bonded to the carbamate. Exposing the carbamate to conditions that remove such protecting group or residual group as described above also results in the removal of the carbamate.

[0461] Furthermore, the aforementioned O-bonded, removable residual / protecting groups can be bonded to the nitrogen on the nucleic acid base using an intermediate structure such as a carbamate (e.g., Z). For example, the carbamate moiety can be bonded to the nitrogen on the nucleic acid base, and the aforementioned group / moiety (-L1 or -L1-L2) can be bonded to the oxygen of the carbamate. Also, under conditions suitable for the removal of such protecting or residual groups from the carbamate, the release of the carbamate from the nitrogen of the nucleic acid base occurs.

[0462] An example of the corresponding structure of the removable portion attached to the carbamate bonded to the nitrogen of the nucleic acid base is shown below.

[0463] An exemplary Z1-L1-L2 structure of a complex having a linker containing a sulfonyl group bonded to a nitrogen atom via a carbamate group is shown below. Such a Z1-L1-L2 structure can be used for synthesis in nucleotides with residual groups (after linker cleavage) or protected nucleotides. An exemplary complex having a sulfonyl group (which is retained as a residual group on the nucleotide after linker cleavage and can be removed) bonded to the nitrogen on the nucleic acid base (via a carbamate) is shown below. TIFF2026522421000103.tif125165

[0464] After linker cleavage, the removal of any remaining residual groups can be achieved by exposing the nucleotides containing the residual groups to a suitable base.

[0465] An exemplary synthesis of N-linked carbamate sulfone nucleotides is shown in Scheme 5. Scheme 5 TIFF2026522421000104.tif89165

[0466] While we do not wish to be bound by theory, an exemplary reaction mechanism for removing the aforementioned residual and protecting groups is beta-elimination, as shown below. TIFF2026522421000105.tif42165

[0467] A similar mechanism applies to protecting or residual groups attached to carbamates that have electron-withdrawing groups. TIFF2026522421000106.tif42165

[0468] For example, the cyanoethyl residual group / protecting group attached to the carbamate is as follows: Therefore, suitable residual or protecting groups include N-linked carbamates bonded to an L1-L2 structure having an electron-drawing group.

[0469] An exemplary Z1-L1-L2 structure of a complex having a linker containing a thioether group bonded to a nitrogen atom via a carbamate group is shown below. Such a Z1-L1-L2 structure can be used for synthesis with nucleotides having residual groups (after linker cleavage) or protected nucleotides. An exemplary complex having a thioether group (which is retained as a residual group on the nucleotide after linker cleavage and can be removed) bonded to the nitrogen on the nucleic acid base (via a carbamate) is shown below. TIFF2026522421000108.tif125165

[0470] After linker cleavage, the removal of remaining residual groups can be achieved by exposing the nucleotides containing the residual groups to an oxidizing agent, followed by exposure to basic conditions.

[0471] An exemplary synthesis of N-linked carbamate thioether nucleotides is shown in Scheme 6. Scheme 6 TIFF2026522421000109.tif57165

[0472] In some embodiments, the linker contains an acyl group bonded to the nitrogen of the nucleic acid base. In some embodiments, the residual group or protecting group contains an acyl group bonded to the nitrogen of the nucleic acid base. In some embodiments, in the structure Z-L1-L2, Z is an acyl group. In some embodiments, the residual group or protecting group containing an acyl group is removed by exposure to a suitable nucleophile or base. In some embodiments, the residual group contains a group that is removed by exposure to a suitable nucleophile or base. In some embodiments, the acyl group is a carbamate or an amide. An exemplary reaction mechanism for removing a residual group or protecting group containing an acyl group (a carbamate if X is O, or an amide if X is C) by a nucleophile is shown below. TIFF2026522421000110.tif47165

[0473] An exemplary reaction mechanism for removing a residual group or protecting group containing an acyl group (a carbamate when X is O, and an amide when X is C) with a base is shown below. TIFF2026522421000111.tif37165

[0474] Therefore, in some embodiments, when Z is an amide group or a carbamate, a number of suitable L1-L2 structures can be used that are suitable for removing the residual or leaving group from the nitrogen of the nucleotide by exposure to a nucleophile or a base.

[0475] Intramolecular cyclization In some embodiments, the linker contains a carbamate group. In some embodiments, the residual group contains a carbamate group. In some embodiments, the residual group contains a carbamate group that is removed by exposure to a suitable base. In some embodiments, the residual group contains a group that is removed by exposure to a suitable base. In some embodiments, the preferred base is a strong base. In some embodiments, the preferred base is a hydroxide with a suitable counterion. In some embodiments, the preferred base is selected from the group consisting of NaOH, NH4OH, KOH, KOtBu, NaOMe, and KOMe.

[0476] In some embodiments, -ZL 1 -L 2 -R 1 The following is true: In formula TIFF2026522421000112.tif22165, X is O, CH2, or NH, n is 1 or 2, and R 1 is hydrogen, -OH, -N(R b )2, or -SH, where each R b C is independently hydrogen or may be substituted. 1-6 It is an alkyl group, and the hydrogen atoms bonded to the carbon atoms between the carbonyl group and the R1 group are, respectively, C 1-3 It may be substituted with an alkyl group.

[0477] In some embodiments, -ZL 1 -L 2 -R 1 teeth, Selected from the group consisting of TIFF2026522421000113.tif27165.

[0478] While not bound by any particular theory, as suggested by the results of Example 11, exposure of a protected nucleic acid base having a residual group containing a carbamate or amide group linked to an alkyl group of a preferred length results in cyclization and subsequent removal of the residual group. This is preferable to the removal mechanisms by nucleophiles or bases described above.

[0479] The reaction mechanism for intramolecular cyclization of N-bonded carbamates with L1-L2 residual groups, which are suitable for intramolecular cyclization, is shown below. TIFF2026522421000114.tif37165

[0480] Soap-in-Gold effect While not bound by any particular theory, as the results of Example 12 suggest, the removal of residual groups by intramolecular cyclization can be improved by utilizing the soap-in-gold effect. For example, L 1 By introducing substituents, the rate of removal of residual groups can be increased. For example, residual groups containing a single methyl substituent can be removed faster than unsubstituted residual groups. Exemplary intramolecular cyclization reactions for the removal of residual groups or protecting groups containing unsubstituted and substituted alkyl groups are shown in Figure 5A (alkyl group attached to an amide group bonded to the nitrogen on the nucleic acid base) and Figure 5B (alkyl group attached to a carbamate group bonded to the nitrogen on the nucleic acid base). As shown in Example 12, the elimination of residual groups by cyclization can be further accelerated by gem-dimethyl substitution (soap-in-gold effect). Therefore, the addition and removal of methyl substituents can be varied depending on the desired stability and required removal efficiency, as this affects the reaction rate.

[0481] In some embodiments, alkyl substituents are added to improve the efficiency of removing residual groups or protected nucleic acid bases. 1 Aside from the adjacent carbon, the carbonyl group and R 1 It can be attached to carbon groups at any position between the existing groups.

[0482] In some embodiments, -ZL 1 -L 2 -R 1 teeth, The selection is made from the group consisting of TIFF2026522421000115.tif47165.

[0483] In some embodiments, the complex is Selected from the group consisting of TIFF2026522421000116.tif78165.

[0484] In some embodiments, the nucleic acid base having a residual group is Selected from the group consisting of TIFF2026522421000117.tif63165.

[0485] Synthesis scheme In some embodiments, complexes or nucleotides containing a carbamate or amide (Z) bound to the nitrogen of a nucleotide and linked to L1-L2-R1 or L1-L2-L3 can be prepared as outlined below. Scheme 7: Synthesis of N6 carbamate sulfide A TIFF2026522421000118.tif58165 Scheme 8: Synthesis of N4 Carbamate Sulfide C TIFF2026522421000119.tif68165 Scheme 9: Synthesis of N4 carbamate ethyl C, N4 carbamate (methyl) ethyl C, and N4 carbamate (dimethyl) ethyl C TIFF2026522421000120.tif83165 Scheme 10: Synthesis of N6 carbamate ethyl A, N6 carbamate (methyl) ethyl A, and N6 carbamate (dimethyl) ethyl A TIFF2026522421000121.tif125165

[0486] General protocol for carbamate formation: 3',5'-diTBS protected nucleosides (dA and dC) (1 equivalent) were dried by repeated co-evaporation with pyridine and toluene, and then dissolved in 1,2-dichloroethane (1 M solution). CDI (1.6 equivalents) was added to the solution, and the mixture was stirred under reflux for 20 hours. Alcohol (2 equivalents) dissolved in 1,2-dichloroethane was added, and the solution was stirred under reflux for 20 hours (3 to 5 hours for C-nucleosides). The solution was cooled to room temperature and washed with saturated NaHCO3 aqueous solution. The aqueous solution was then back-extracted with CH2Cl2, and the collected organic solution was dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel column chromatography with a 0-50% siRNA / hexane gradient to obtain carbamates 2 and 7.

[0487] General protocol for deprotection of the benzyl group (compounds 23 / 24 / 25): 10% Pd / C (10% w / w) was added to a solution of compounds 20 / 21 / 22 (1 mmol) in MeOH (3 mL). The internal air was replaced by three vacuum / H2 cycles using an H2 balloon. The reaction mixture was stirred at room temperature until complete consumption of the starting material was confirmed by monitoring with thin-layer chromatography. Next, the reaction mixture was passed through a Celite pad or membrane filter using siRNA to remove Pd / C. The filtrate was concentrated under vacuum to obtain the deprotection product.

[0488] General protocol for esterification reaction (compounds 3 / 8 / 26 / 27 / 28): To a solution of compound 2 / 7 (1 equivalent) in anhydrous DCM (0.2 M), DMAP (0.6 equivalents) and Fmoc amino acid (1.2 equivalents) were added at room temperature under an inert atmosphere. The reaction mixture was cooled to 0°C, and then EDC·HCl (1.2 equivalents) was slowly added. The reaction mixture was stirred at room temperature for 16 hours. The reaction solution was then diluted further with DCM and extracted with water. The aqueous layer was then washed with more DCM (2x). The combined organic layers were then washed with saturated ammonium bicarbonate, dried over Na2SO4, and concentrated. The crude product was purified by silica gel column chromatography with a 10–45% siRNA / hexane gradient.

[0489] General procedure for TBS deprotection (compounds 4 / 9 / 14 / 15 / 16 / 29 / 30 / 31): To a 0.1 M solution of the protected nucleoside (3 / 8) in anhydrous THF at 0°C, 3HF-TEA (10 equivalents) was added dropwise. The mixture was stirred for 16 hours while warming to room temperature. A few drops of MeOH were added to quench the reaction, and the solvent was removed under reduced pressure. The residue was diluted with DCM and washed with water. The aqueous layer was extracted again with DCM (1x). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated. The compounds were purified by silica gel column chromatography with a 1-12% MeOH / DCM gradient. All compounds were obtained as white foamy solids.

[0490] General protocol for the synthesis of triphosphates (compounds 5 and 10): Nucleoside analogs (4 / 9 / 14 / 15 / 16, 50 mg, 1 equivalent) were placed in a 10 mL round-bottom flask with a stirring bar. Tetrabutylammonium pyrophosphate was placed in a separate 5 mL conical tube. These two flasks were placed in a vacuum desiccator with P2O5 and dried under vacuum for at least 16 hours. Furthermore, the molecular sieves and three small round-bottom flasks were placed in a drying oven for at least 16 hours. Molecular sieves were placed in two of the small flasks removed from the oven and flame-activated under vacuum. While the molecular sieves cooled, another small flask was attached to a Hickman distillation apparatus and flame-dried. During cooling, the first two flasks were filled with nitrogen. Trimethyl phosphate and tributylamine were then placed on the molecular sieves in the first two flasks to dry. POCl3 was then freshly distilled using the Hickman distillation apparatus. The vacuum desiccator was purged with N2 gas, and the flask inside was then transferred to a nitrogen balloon or Schlenk line. Trimethyl phosphate (40 equivalents) was added to the nucleoside, and the mixture was cooled to -5°C. To this nucleoside mixture, anhydrous tributylamine (3 equivalents), followed by POCl3 (2.1 equivalents), was slowly added using a microsyringe. The combined mixture was stirred at -5°C for 45 minutes. After 45 minutes, the reaction mixture was treated with a mixture of tributylamine pyrophosphate (5 equivalents, 0.5 M in anhydrous acetonitrile) and tributylamine (6 equivalents). After 1 hour, the mixture was treated with triethylammonium bicarbonate (0.5 M, 1:2 of the total reaction volume) and stirred at room temperature for 1 hour.

[0491] Fmoc deprotection (compounds 5 / 10 / 32 / 33 / 34): Next, this reaction mixture was further treated with N-methylpiperidine (1 / 5 of the total reaction volume), stirred at room temperature for 90 minutes, and then extracted with dichloromethane (2X). The aqueous layer was then subjected to reverse-phase HPLC (0.1M triethylammonium acetate buffer / acetonitrile, 4-47%, 0-15 mins, flow rate 5 ml). -1The product was purified by [method / method]. The fraction containing the product was pooled and freeze-dried to obtain the desired product as a triethylammonium salt. The resulting solid was reconstituted in RNase-free DI water for further experiments.

[0492] Boc deprotection (compounds 17 / 18 / 19): The reaction was quenched with triethylammonium bicarbonate (0.5 M, 1:2 of the total reaction volume), stirred at room temperature for 1 hour, and the reaction mixture was extracted with dichloromethane (2X). The aqueous layer was then subjected to reverse-phase HPLC (0.1 M triethylammonium acetate buffer / acetonitrile, 4-47%, 0-15 mins, flow rate 5 ml). -1 The triphosphate was purified by [method]. The fraction containing triphosphate was pooled and freeze-dried to obtain triphosphate with linker-amine-Boc. This was divided into four Eppendorf tubes. MeOH (10 μL) and TFA (20 μL) were added to each Eppendorf tube. After vortexing, the mixture was held at room temperature for 2 minutes and Et2O (500 μL) was added. After vortexing, the mixture was cooled at -20°C for 10 minutes. After centrifugation (10,000 rpm, 5 minutes), the supernatant was decanted. This ether washing procedure was repeated twice. The resulting solid was reconstituted in RNase-free DI water for further experiments.

[0493] For each nucleotide, an exemplary reaction scheme for preparing a complex or nucleotide containing a removable residual group or protecting group attached to the nitrogen on the nucleic acid base is shown below. Scheme 11: A general synthetic scheme for obtaining nucleotides modified with cleavable amides and carbamates bonded to the N2 position of G: TIFF2026522421000122.tif104165

[0494] Scheme 12: A general synthetic scheme for obtaining nucleotides modified with cleavable amides and carbamates bonded to the N1 position of G: TIFF2026522421000123.tif145165

[0495] Scheme 13: A general synthetic scheme for obtaining nucleotides modified with cleavable amides and carbamates bonded to the N6 position of A: TIFF2026522421000124.tif89165

[0496] Scheme 14: A general synthetic scheme for obtaining nucleotides modified with cleavable amides and carbamates bonded to the N4 position of C: TIFF2026522421000125.tif89165

[0497] Scheme 15: A general synthetic scheme for obtaining nucleotides modified with cleavable amides and carbamates bonded to the N3 position of U / T: TIFF2026522421000126.tif94165

[0498] Light-cuttable protective group In some embodiments, a photocleavable group is used as a protecting or residual group that can be removed from the nucleic acid base. The photocleavable group can be removed by exposure to light of an appropriate wavelength, leaving the native nucleotide. In some embodiments, the wavelength of light is ultraviolet (UV). In some embodiments, the linker of the complex bonded to oxygen or nitrogen on the nucleic acid base contains a photocleavable group. In some embodiments, the residual group after linker cleavage contains a photocleavable group. In some embodiments, the nucleotide has a protecting group containing a photocleavable group.

[0499] In some embodiments, L 1 It contains a light-cleavable group. In some embodiments, L 1 It contains a 2-nitrobenzyl group which may be substituted. In some embodiments, L 1 It contains a 5-methoxy-2-nitrobenzyl group. In some embodiments, L 1 The following is true: TIFF2026522421000127.tif32165, each R aThis is independently selected from the group consisting of halogens, -Me, and -OMe.

[0500] In some embodiments, L 1 The following applies: TIFF2026522421000128.tif42165

[0501] In some embodiments, a complex comprising nucleotides bound to a photocleavable group is represented as follows: In formula TIFF2026522421000129.tif32165, the linker is attached to the nucleotide via the oxygen or nitrogen of the nucleic acid base.

[0502] In some embodiments, a protected nucleotide or a nucleotide having a residual group, which includes a nucleotide bonded to a photocleavable group, is represented as follows: In formula TIFF2026522421000130.tif32165, the nitrobenzyl group is attached to the nucleotide via either the oxygen or nitrogen of the nucleic acid base.

[0503] An exemplary Z1-L1-L2 structure of a complex having a linker containing a nitrobenzyl photocleavable group is shown below. Such a Z1-L1-L2 structure can be used for synthesis with nucleotides having residual groups (after linker cleavage) or protected nucleotides. An exemplary complex having a photocleavable group bonded to the oxygen on the nucleic acid base (which is retained as a residual group on the nucleotide after linker cleavage and can be removed) is shown below. TIFF2026522421000131.tif99165TIFF2026522421000132.tif194165TIFF2026522421000133.tif135165

[0504] In some embodiments, a protected nucleotide containing a photocleavable group or a nucleotide having a residual group is, Selected from the group consisting of TIFF2026522421000134.tif99165.

[0505] In some embodiments, protected nucleic acid bases containing photocleavable protecting or residual groups bonded to oxygen on the nucleic acid base are Selected from the group consisting of TIFF2026522421000135.tif83165, In the formula, R 1 It is selected from the group consisting of hydrogen, -OH, -N(Rb)2, and -SH. Each Rb is independently a hydrogenated or possibly substituted C1-6 alkyl group.

[0506] In some embodiments, the protected nucleic acid base is The selection is made from the group consisting of TIFF2026522421000136.tif190165 and TIFF2026522421000137.tif221165.

[0507] In some embodiments, the disclosure includes a method for preparing polynucleotides, the method including the removal of residual or protecting groups bound to nucleic acid bases by photolysis. In some embodiments, the photolysis includes exposing the polynucleotides to ultraviolet light. In some embodiments, the photolysis includes exposing the polynucleotides to light with a wavelength of about 365 nm.

[0508] For example, the removal of residual groups can be achieved by exposing nucleic acid bases to light having a wavelength of approximately 365 nm, as follows: TIFF2026522421000138.tif53165

[0509] In some embodiments, complexes or nucleotides containing photocleavable groups can be prepared as outlined in Scheme 16. Scheme 16 TIFF2026522421000139.tif48165

[0510] In some embodiments, the photocleavable linker can also be bound to the nitrogen of a nucleic acid base. An exemplary scheme for preparing an N-linked photocleavable group bound to adenine is outlined in Scheme 17. Scheme 17 TIFF2026522421000140.tif48165

[0511] An example of a complex containing an N-linked carbamate and a photocleavable nitrobenzyl protecting group is shown below. When L3 is cleaved, the nucleotide will have an N-linked residual group that can be cleaved by photolysis. TIFF2026522421000141.tif119165

[0512] In some embodiments, the linker of the complex may be bonded to oxygen or nitrogen on the nucleic acid base. In certain embodiments, when the linker of the complex bonded to oxygen or nitrogen on the nucleic acid base is cleaved, residual groups remain, which may also act as protecting groups that inhibit the formation of secondary structures. In some embodiments, residual groups are removed using chemical or photodegradation conditions that can remove one or more protecting groups from the protected nucleic acid base.

[0513] Linker binding to polymerase In some embodiments, L3 includes a biobinding group suitable for binding L3 to polymerase.

[0514] In some embodiments, the biobinding group is an N-hydroxysuccinimide (NHS) group. In some embodiments, the biobinding group is a maleimide group. Therefore, the linker may be covalently bonded to the polymerase by the reaction of the maleimide group with a cysteine ​​residue of the polymerase.

[0515] In some embodiments, the polymerase may be operably bound to a linker moiety. Such linker moieties may include covalent or non-covalent bonds, amino acid tags (e.g., poly-amino acid tags, poly-His tags, 6His tags (SEQ ID NO: 53)), compounds (e.g., polyethylene glycol), protein-protein binding pairs (e.g., biotin-avidin), affinity couplings, capture probes, or any combination thereof. The linker moiety may be separate from the polymerase variant or may be part of the polymerase variant.

[0516] Phosphatase treatment of the complex to reduce insertion In particular, this disclosure provides polymerase-nucleotide complexes. As will be apparent to those skilled in the art, there are various challenges associated with the accurate and precise synthesis of polynucleotides. For example, polymerases can be prone to errors in their catalytic action of adding nucleotides by covalent bonding. Therefore, when polymerase-nucleotide complexes are used in controlled, stepwise nucleic acid synthesis, the addition of two or more nucleotides per step (e.g., insertions or non-terminations) may occur. This specification provides techniques that overcome such challenges by combining such complexes with phosphatases (e.g., supplying polymerase-nucleotide complexes in the presence of phosphatases). Such techniques help reduce errors and achieve more accurate and precise stepwise addition compared to the aforementioned synthesis methods (e.g., those performed in the absence of phosphatases).

[0517] In some embodiments, the complex is supplied in the presence of a phosphatase. In some embodiments, the disclosure provides a complex reagent comprising a plurality of polymerase-nucleotide complexes, in which the polymerase and nucleotides are linked via a linker. In some embodiments, the linker is cleavable.

[0518] In some embodiments, the complex reagent is present in the presence of a phosphatase.

[0519] In some embodiments, the polymerase-nucleotide complex is combined with a template (e.g., a starting oligonucleotide or a first oligonucleotide) in the presence of a phosphatase.

[0520] A typical process for the stepwise synthesis of polynucleotides involves the stepwise addition of individual nucleotides to a starting oligonucleotide (i.e., the initial oligonucleotide) through a series of repeated steps. For example, in some embodiments, the steps include attaching a polymerase-nucleotide complex to the oligonucleotide, covalently attaching a nucleotide to the 3' end of the oligonucleotide by catalytic action of the polymerase, and cleaving the polymerase from the attached nucleotide. These steps can be repeated until the desired elongated polynucleotide is synthesized, resulting in an elongated polynucleotide that is at least one nucleotide longer than the polynucleotide before the steps were repeated one or more times.

[0521] In particular, this specification provides methods for nucleic acid synthesis. In some embodiments, the nucleic acid synthesis method includes the step of contacting (e.g., incubating) a complex reagent containing a polymerase-nucleotide complex (e.g., multiple polymerase-nucleotide complexes) in the presence of one or more phosphatases. In some embodiments, the multiple nucleotides are the same nucleotide (e.g., A, G, T, or C). In some embodiments, the multiple nucleotides are different nucleotides (e.g., A, G, T, and / or C). In some such embodiments, the synthesis performed in the presence of a phosphatase is improved in one or more respects compared to the same synthesis in the absence of a phosphatase (e.g., more precise, more efficient, and more accurate).

[0522] In some embodiments, synthesis carried out in the presence of a phosphatase prevents the addition of unshielded nucleotides to nucleic acids. The method provided herein includes a step of contacting (e.g., incubating) a complex reagent containing multiple polymerase-nucleotide complexes with a phosphatase, and using the polymerase-nucleotide complexes in nucleic acid synthesis reduces the proportion of processes that result in the addition of two or more nucleotides per step (e.g., unterminated processes leading to additional nucleotide insertions) compared to synthesis without a phosphatase or without processing the complex reagent with a phosphatase.

[0523] Achieving precise single-nucleotide addition at each step of stepwise nucleic acid synthesis is essential for the accurate synthesis of longer oligonucleotides. Performing such additions with accuracy and precision remains a challenge in the industry. For example, stepwise nucleic acid synthesis using polymerase-nucleotide complexes is susceptible to insertions and / or non-terminations, which can result in the addition of two or more nucleotides to a nucleic acid in a single step of repeated nucleotide elongation.

[0524] Unshielded nucleotides are either sterically unshielded or only partially sterically shielded by the polymerase that anchors them to phosphatase cleavage at their 5' phosphate. During oligonucleotide synthesis, polymerases can catalyze errors that lead to the covalent addition of unshielded nucleotides, and therefore, using polymerase-nucleotide complexes in nucleic acid synthesis may result in the addition of two or more nucleotides per step (e.g., insertion or non-termination). The techniques provided herein overcome this challenge and help achieve accurate and precise stepwise addition with fewer errors compared to the aforementioned synthetic methods.

[0525] In some embodiments, non-termination may occur when adding an unshielded nucleotide having an uncleaved 5' phosphate to an oligonucleotide. As shown herein, in some embodiments, a phosphatase hydrolyzes the 5' phosphate (e.g., terminal 5' phosphate) of the nucleotide (e.g., nucleotide triphosphate). In some embodiments, the terminal 5' phosphate is located on the α-phosphate, β-phosphate, χ-phosphate, δ-phosphate, ε-phosphate, φ-phosphate, or γ-phosphate of the nucleotide. In some embodiments, the phosphatases disclosed herein hydrolyze the 5' phosphate (e.g., terminal 5' phosphate) of the nucleotide or free nucleotide in the polymerase-nucleotide complex. In some embodiments, the phosphatases disclosed herein hydrolyze the 5' phosphate of the nucleotide (e.g., one or more 5' terminal phosphates of nucleotides in multiple polymerase-nucleotide complexes), so that the hydrolyzed nucleotide is prevented from being added to the nucleic acid during oligonucleotide synthesis. In...

Claims

1. A template-independent de novo synthesis method for long single-stranded polynucleotides, a) To provide a substrate, wherein the substrate contains a plurality of free hydroxyl groups that are bonded to the substrate and suitable for nucleotide bonding, b) Bonding the nucleotide attached to the protecting group to the plurality of free hydroxyl groups, c) Removing the protecting group from the bound nucleotide, d) Repeating steps (b) and (c) according to a predetermined nucleotide sequence (e.g., a reference sequence) to obtain a plurality of de novo-synthesized polynucleotides of at least 500 nucleotides in length, The error rate observed for each bond is less than 1% compared to the predetermined polynucleotide sequence. The aforementioned method.

2. The method according to claim 1, wherein the de novo-synthesized long polynucleotide is at least 600 nucleotides long, at least 700 nucleotides long, at least 800 nucleotides long, at least 900 nucleotides long, at least 1000 nucleotides long, at least 1100 nucleotides long, at least 1200 nucleotides long, at least 1300 nucleotides long, at least 1400 nucleotides long, at least 1500 nucleotides long, at least 1600 nucleotides long, at least 1700 nucleotides long, at least 1800 nucleotides long, at least 1900 nucleotides long, or at least 2000 nucleotides long.

3. The method according to claim 1, wherein the de novo-synthesized long polynucleotide is 500 to 1000 nucleotides long, 500 to 1500 nucleotides long, 500 to 2000 nucleotides long, or 500 to 2500 nucleotides long.

4. The method according to claim 1, wherein the error rate observed for each of the aforementioned connections 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.

5. The method according to claim 1, wherein the coupling step is performed in less than 120 seconds, less than 90 seconds, less than 60 seconds, less than 50 seconds, 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.

6. The method according to claim 1, wherein the template-independent polynucleotide synthesis is carried out 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.

7. The method according to claim 1, wherein the template-independent polynucleotide synthesis is performed at a rate of 5 to 25 nucleotides per hour, 10 to 30 nucleotides per hour, 15 to 45 nucleotides per hour, or 20 to 60 nucleotides per hour.

8. The plurality of de novo synthesized polynucleotides on the substrate include 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 10×10 ５ polynucleotides, at least 10×10 ６ polynucleotides, at least 10×10 ７ polynucleotides, at least 10×10 ８ polynucleotides, at least 10×10 ９ polynucleotides, at least 10×10 １０ polynucleotides, at least 10×10 １１ polynucleotides, at least 10×10 １２ polynucleotides, at least 10×10 １３ polynucleotides, or at least 10×10 １４ polynucleotides, and the method according to claim 1.

9. The method according to claim 1, wherein the free hydroxyl group is located at the end of a plurality of starting oligonucleotides or elongated polynucleotides bonded to the substrate.

10. The method according to claim 9, wherein the starting oligonucleotide includes a single-stranded region at its 3' end.

11. The method according to claim 9, wherein the starting oligonucleotide is hybridized to the oligonucleotide bound to the substrate.

12. The method according to claim 9, wherein the starting oligonucleotide is covalently bonded to the substrate.

13. The method according to claim 1, wherein the bonding of the nucleotides is carried out by an enzyme.

14. The method according to claim 1, wherein the bonding of the nucleotides is carried out by the catalytic action of polymerase.

15. The method according to claim 14, wherein the polymerase is a template-independent polymerase.

16. The method according to claim 15, wherein the template-independent polymerase is covalently bonded to the nucleotide.

17. The method according to claim 15 or 16, wherein the template-independent polymerase is terminal deoxynucleotidyltransferase (TdT) or a variant thereof.

18. The method according to claim 14, wherein the polymerase is RNA polymerase.

19. The method according to claim 1, wherein the protecting group is a template-independent polymerase bonded to the nucleotide.

20. The method according to claim 19, wherein the removal of the protecting group comprises cleaving a linker that binds the nucleotide to the template-independent polymerase.

21. The method according to claim 1, wherein the protecting group is a 3'-O-protecting group.

22. The method according to claim 21, wherein the removal of the protecting group includes removing the 3'-O-protecting group from the nucleotide in order to leave a free 3'-hydroxyl group.

23. The method according to claim 1, wherein the protecting group is a 2' or 3' modification of the nucleotide.

24. The method according to claim 23, wherein the 2' modification is selected from the group consisting of -H, -OH, -F, -OMe, -N3, -NH2, and -Ara.

25. The 3' modification is -H, -OH, -OCH ２ N ３ , -ONH ２ The method according to claim 23, selected from the group consisting of , and -O allyl.

26. The method according to claim 1, wherein the protecting group is a reversible terminator.

27. The method according to claim 1, wherein 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%.

28. The method according to claim 1 or 27, wherein 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%.

29. The method according to claim 1, wherein the binding is carried out in the presence of a phosphatase.

30. The method according to claim 29, wherein the phosphatase is an inorganic pyrophosphatase.

31. The method according to claim 1, wherein the nucleotide bonded to the protecting group is a nucleotide-polymerase complex, and the complex is treated with a phosphatase.

32. The method according to claim 1, wherein the bonding is carried out in the presence of a divalent cation, and the total concentration of divalent cations present in the reaction volume of the bonding is about 500 μM or less.

33. The method according to claim 32, wherein the total concentration of divalent cations present in the reaction volume is about 250 μM or less, about 125 μM or less, or about 50 μM or less.

34. The divalent cation present at the highest concentration in the reaction volume is cobalt (Co ２＋ ) or zinc (Zn ２＋ The method according to claim 32, which is as follows.

35. At least one of the divalent cations is Mg ２＋ Ca ２＋ , Sr ２＋ Ba ２＋ Mn ２＋ Co ２＋ Fe ２＋ Ni ２＋ ,Cd ２＋ , and Zn ２＋ The method according to claim 32, or a combination thereof.

36. The aforementioned bonding reaction is carried out by Mg ２＋ The method according to claim 32, which is carried out in the absence of [the specified substance].

37. The method according to claim 1, wherein the nucleotide comprises one or more modifications to the hydrogen bond-forming N or O on the nucleic acid base.

38. The method according to claim 1, wherein the bonded nucleotide comprises one or more alkylated nucleic acid bases after the removal of the protecting group.

39. The method according to claim 38, further comprising contacting the de novo-synthesized polynucleotide with an alkyltransferase.

40. The method according to claim 39, wherein the alkyltransferase belongs to EC 2.1.1.

63.

41. The method according to claim 39, wherein the alkyltransferase is selected from the alkyltransferases listed in Table 1 or Table 2.

42. The alkyltransferase is O ６ - The method according to claim 39, wherein the enzyme is alkylguanine DNA alkyltransferase.

43. The method according to claim 39, wherein the alkyltransferase is AlkB.

44. The alkylated nucleic acid bases are as follows: It is expressed by, in the formula, X is -C(R ２ ) = or -N =, R １ However, C １－６ Alkyl, C ２－６ Alkenil, C １－６ Alkinyl and -(CH ２ ) ０－３ Selected from the group consisting of Ph, where R １ However, 1 to 6 R １ａ It may also be replaced with Each R １ａ However, independently, halogen, C １－６ Alkyl, -(CH ２ ) ０－３ OR １ｂ , -NO ２ , -N ３ , -OPO ２ OH, and -(CH ２ ) ０－３ NHR １ｂ Selected from, Each R １ｂ However, independently, hydrogen, C １－６ Alkyl, -C(O)(C １－６ Alkyl), C １－６ Haloalkyl, -C(O)(C １－６ Haloalkyl), and -CH ２ Selected from OAc, R ２ However, hydrogen may be substituted. １－４ Selected from the group consisting of alkyl chains, where 1-2 methylene units are independently -O-, -N(R ａ )-, -C(O)-, -S-, -S(O)-, -S(O) ２ -, or it may be replaced with phenylene, R ２ However, 1 to 6 R ２ａ It may also be replaced with Each R ２ａ However, independently, halogen, C １－６ Alkyl, -(CH ２ ) ０－３ OR ２ｂ , -NO ２ , -N ３ , -OPO ２ OH, and -(CH ２ ) ０－３ NHR ２ｂ Selected from, Each R ２ｂ However, independently, hydrogen and C １－６ Selected from alkyl groups, The method according to claim 38.

45. R １ However, C １－４ The method according to claim 38, wherein the alkyl group is used.

46. R １ The method according to claim 39, wherein the substance is selected from the group consisting of methyl, ethyl, n-propyl, and n-butyl.

47. R １ but, The method according to claim 38, selected from the group consisting of the following.

48. R １ａ However, -OR １ｂ The method according to claim 47.

49. R １ｂ The method according to claim 48, wherein the hydrogen is...

50. The alkylated nucleic acid base in the polynucleotide is The method according to claim 38, selected from the group consisting of the following.

51. The method according to claim 1, wherein the bonded nucleotide, after removal of the protecting group, includes one or more modifications to the base-pairing nitrogen or oxygen on the nucleic acid base.

52. The aforementioned bonded nucleotides are (B) below: It is expressed by, in the formula, R is ribose polyphosphate or deoxyribose polyphosphate, Y is a nucleic acid base, L-R １ However, it is a protecting group, L is bonded to the base-pairing nitrogen or oxygen of the nucleic acid base, R １ However, hydrogen, -OH, -N(R ｂ ) ２ Selected from the group consisting of , and -SH, where each R ｂ However, C may be hydrogenated or substituted independently. １－６ It is alkyl. The method according to claim 1.

53. L is -Z-L １ -L ２ - and 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 However, Selected from the group consisting of, L １ However, 1 to 4 R Ｌ It may also be replaced with Each R Ｌ is independently selected from the group consisting of halogen, hydroxyl, oxo, and optionally substituted C １ -C ３ -alkyl, where two Rs １ may together with intervening atom(s) form a 3- to 6-membered carbocyclic ring, L ２ However, C may be bonded or substituted. １－１２ Alkylene chain, C ４ -C ２０ Polyethylene glycol, which may be substituted C ２－１２ Alkenylene chain and optionally substituted C ２－１２ Selected from a group consisting of alkynylene chains, where 1 to 6 methylene units are independently -O-, -N(R ｂ )-, -C(O)-, -S-, -S(O)-, -S(O) ２ - or it may be replaced with phenylene, W is -O-, -S-, -S(O) ２ -, and -N(R ｂ Selected from the group consisting of ) Each R ａ However, it is halogen, -Me, or -OMe, Each R ｂ However, independently, hydrogen or C １－６ It is alkyl, n is 1 or 2, L １ Z and Z cannot be bonded at the same time. The method according to claim 52.

54. The method according to claim 53, wherein Z is a bond when L is bonded to a base-pairing oxygen of the nucleic acid base.

55. When L is bonded to the base-pairing nitrogen of the nucleic acid base, Z is -C(O)-, -C(O)CH ２ -, -C(O)C(R Ｌ ) ２ -, -C(O)CH(R Ｌ The method according to claim 53, selected from the group consisting of -, -C(O)O-, and -C(O)N(H)-.

56. L １ but, And, L １ However, 1 to 4 R Ｌ It may also be replaced with n is 1 or 2, W is -O-, -S-, -S(O) ２ -, and -N(R ｂ Selected from the group consisting of ) The method according to any one of claims 53 to 55.

57. L １ but, Selected from the group consisting of, L １ However, 1 to 4 R Ｌ The method according to claim 56, which may be replaced by the following.

58. Each R Ｌ However, C may be substituted independently. １ -C ３ It is alkyl, and here there are two R １ The method according to any one of claims 53 to 57, wherein intervening atoms (or more) may form a 3- to 6-membered carbocyclyl ring.

59. R Ｌ The method according to claim 58, wherein the methyl molecule may be substituted.

60. L １ but, The method according to any one of claims 53 to 57, selected from the group consisting of the following.

61. The method according to any one of claims 53 to 60, wherein Z is -C(O)O-.

62. The method according to any one of claims 53 to 60, wherein Z is -C(O)N(H)-.

63. The method according to any one of claims 53 to 60, wherein Z is a combination.

64. The method according to any one of claims 53 to 60, wherein Z is -C(O)-.

65. -Z-L １ -8 ２ -2 １ が、 The method according to any one of claims 53 to 60, selected from the group consisting of the following.

66. L ２ However, C may be substituted. １－１２ This is an alkylene chain, where 1 to 6 methylene units are independently -O-, -N(R) ｂ )-, -C(O)-, -S-, -S(O)-, -S(O) ２ - The method according to any one of claims 53 to 60, which may be replaced with phenylene.

67. L ２ However, C may be substituted. １－１２ The method according to claim 66, wherein the alkylene chain is such that 1 to 6 methylene units are independently replaced by -O-.

68. L ２ However, C may be substituted. ２－６ The method according to claim 67, wherein the alkylene chain is such that 1 to 3 methylene units are independently replaced by -O-.

69. -Z-L １ -8 ２ -2 １ が、 The method according to any one of claims 53 to 60, selected from the group consisting of the following.

70. The method according to claim 1, wherein the bonding includes immersing the substrate in a solution containing the nucleotide and a template-independent polymerase.

71. The method according to claim 1, wherein the protecting group is a polymerase, and the polymerase is bound to the nucleotide via a cleavable linker.

72. The method according to claim 71, wherein the cleavable linker comprises an amino acid ester.

73. The method according to claim 72, wherein the amino acid ester is bonded to an amino acid.

74. The method according to claim 73, wherein the amine group of the amino acid ester is bonded to the amino acid.

75. The method according to claim 74, wherein the cleavable linker comprises a peptide of at least two, at least three, at least four, or at least five amino acids bonded to the amine group of the amino acid ester.

76. The method according to any one of claims 72 to 75, wherein one or more of the amino acids are 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.

77. The method according to any one of claims 72 to 75, wherein the amino acid is glycine, or a plurality of the amino acids include glycine.

78. The method according to any one of claims 72 to 75, wherein the amino acid is an amino acid that does not exist in nature, or a plurality of the amino acids include an amino acid that does not exist in nature.

79. The method according to any one of claims 71 to 78, wherein the cleavable linker is bonded to the alpha-phosphate, sugar, or nucleic acid base of the nucleotide.

80. The aforementioned amino acid ester, Represented by, In the formula, R １ and R １’ However, each can be independently of hydrogen and C which may be substituted. １－６ C may be substituted, either selected from alkyl groups or together with the atom to which they are bonded. ３ -C ７ The method according to any one of claims 72 to 79, wherein a carbocyclic ring may be formed.

81. The aforementioned amino acid ester, The method according to claim 80, represented by a compound selected from the group consisting of the following.

82. The linker has the following structure: Including, in the formula, R １ and R １’ However, each can be independently of hydrogen and C which may be substituted. １－６ C may be substituted, either selected from alkyl groups or together with the atom to which they are bonded. ３ -C ７ It may also form a carbocyclic ring, Each R ２ However, hydrogen, C １－６ Alkyl, phenyl, C １ -C ６ A substituted group independently selected from the group consisting of carbocyclic rings and heterocycles with 3 to 7 members, Each R ３ However, C may be hydrogenated or substituted. １－６ It is alkyl, n is 1, 2, 3, 4, or 5. The method according to any one of claims 72 to 79.

83. R ３ The method according to claim 82, wherein the hydrogen is...

84. R ２ The method according to claim 82 or 83, wherein the hydrogen is...

85. R ２ However, hydrogen, -Me, isopropyl, -sec-butyl, isobutyl, -CH ２ Ph, -CH ２ OH, -CH ２ SH, -CH ２ CH ２ SCH ３ ien-CH ２ COOH, -CH ２ CH ２ COOH, -CH ２ CONH ２ ien-CH ２ CH ２ CONH ２ ien-CH ２ CH ２ ,CH ２ CH ２ NH ２ , The method according to claim 82 or 83, selected from the group consisting of the following.

86. The method according to any one of claims 82 to 85, wherein n is 1.

87. R １ and R １’ However, C may be replaced together with it. ３ -C ７ The method according to any one of claims 82 to 86, wherein a carbocyclic ring is formed.

88. R １ and R １’ However, C may be replaced together with it. ３ The method according to claim 87, wherein a carbocyclic ring is formed.

89. The linker has the following structure: The method according to any one of claims 72 to 79, including the method described in any one of claims 72 to 79.

90. The nucleotide bound to the polymerase has the following structure: Nuc-L1-L2-L3-Pol Including, in the formula, Nuc is a nucleotide, Pol is a polymerase, L1 is the first part of the linker that connects the nucleotide to L2, L2 is as follows: The second part of the linker is represented by the formula, in which, R １ and R １’ However, each C may be substituted independently. １－６ C may be substituted with an atom selected from alkyl or halogen atoms, or together with the atom to which they are bonded. ３ -C ７ It may also form a carbocyclic ring, Each R ２ However, hydrogen, C １－６ Alkyl, phenyl, C １ -C ６ A substituted group independently selected from the group consisting of carbocyclic rings and heterocycles with 3 to 7 members, Each R ３ However, C may be hydrogenated or substituted. １－６ It is alkyl, n is 0, 1, 2, 3, 4, or 5, * indicates the connection point from L2 to L1, and ** indicates the connection point from L2 to L3. L ２ However, it can be cut. L ３ However, pol to L ２ It is a linker that connects to The method according to claim 72.

91. L １ However, C may be bonded or substituted. １－１２ Alkylene chain, C ４ -C ２０ Polyethylene glycol, which may be substituted C ２－１２ Alkenylene chain, and C ２－１２ Selected from the group consisting of alkynylene chains, where L １ The 1 to 6 methylene units are independently -O-, -N(R ｂ )-, -N=C(H)-, -C(O)-, -S-, -S(O)-, -S(O) ２ - The method according to claim 90, which may be replaced with substituted phenylene or substituted cyclopropylene.

92. L １ but, Includes, In the formula, each R ａ However, C may be substituted with halogen, hydroxyl, or cyano compounds independently. １－６ Alkyl and optionally substituted C １－６ The method according to claim 91, selected from the group consisting of alkoxys.

93. L ２ but, The method according to any one of claims 90 to 92, comprising an amino acid ester selected from the group consisting of the following.

94. L ２ but, The method according to claim 93, as represented by [the specified method].

95. L １ The method according to any one of claims 90 to 94, wherein the nucleic acid base of the nucleotide is bonded to the nucleic acid base.

96. L １ The method according to claim 95, wherein the nucleic acid base is bonded to oxygen or nitrogen involved in base pairing.

97. The nucleic acid base is The method according to claim 96, selected from the group consisting of the following.

98. L １ The method according to any one of claims 90 to 94, wherein the nucleotide is bonded to a sugar.

99. L １ The method according to any one of claims 90 to 94, wherein the nucleotide is bonded to the phosphate group.

100. The method according to claim 99, wherein the phosphoric acid is alpha-phosphoric acid.

101. The method according to any one of the prior claims, wherein the nucleotide is ribonucleotide polyphosphate or deoxyribonucleotide polyphosphate.

102. The method according to any one of the prior claims, wherein the nucleotide is selected from the group consisting of adenine, guanine, cytosine, uracil, and thymine.

103. The method according to any one of the prior claims, wherein the polymerase is a template-independent polymerase.

104. The method according to claim 103, wherein the polymerase is TdT.

105. The method according to any one of the prior claims, wherein the linker is cleavable by a protease having esterase activity.

106. The method according to claim 105, wherein the linker is cleavable by proteinase K.

107. The method according to claim 105 or 106, wherein the linker is cleavable at the ester group on L2, and a compound represented by Nuc-L1-OH can be left after the cleavage.

108. A substrate comprising a plurality of linked polynucleotides, each at least 500 nucleotides long, wherein the plurality of polynucleotides are generated by a stepwise template-independent polynucleotide synthesis in which the error rate observed per cycle compared to a predetermined nucleotide sequence is less than 1%.

109. The substrate according to claim 108, wherein the polynucleotide is at least 600 nucleotides long, at least 700 nucleotides long, at least 800 nucleotides long, at least 900 nucleotides long, at least 1000 nucleotides long, at least 1100 nucleotides long, at least 1200 nucleotides long, at least 1300 nucleotides long, at least 1400 nucleotides long, at least 1500 nucleotides long, at least 1600 nucleotides long, at least 1700 nucleotides long, at least 1800 nucleotides long, at least 1900 nucleotides long, or at least 2000 nucleotides long.

110. The method according to claim 108, wherein the polynucleotide is 500 to 1000 nucleotides long, 500 to 1500 nucleotides long, 500 to 2000 nucleotides long, or 500 to 2500 nucleotides long.

111. The method according to claim 108, wherein the recognized 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, compared to the predetermined sequence.

112. The method according to claim 108, wherein the recognized 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 compared to the predetermined sequence.

113. The plurality of bound polynucleotides on the substrate consist of 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, and at least 10 × 10 ５ individual polynucleotides, at least 10 × 10 ６ individual polynucleotides, at least 10 × 10 ７ individual polynucleotides, at least 10 × 10 ８ individual polynucleotides, at least 10 × 10 ９ individual polynucleotides, at least 10 × 10 １０ individual polynucleotides, at least 10 × 10 １１ individual polynucleotides, at least 10 × 10 １２ individual polynucleotides, at least 10 × 10 １３ 10 polynucleotides, or at least 10 × 10 １４ The method according to claim 108, comprising 1 polynucleotide.

114. The method according to claim 108, wherein the polynucleotide comprises one or more nucleotides having modifications to hydrogen bond-forming N or O on the nucleic acid base.

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