Nucleotides as substrates for base modification of tdt-based enzymatic nucleic acids

Abstract

The disclosure herein includes methods and compositions for nucleic acid synthesis using terminal deoxynucleotidyl transferase with a plurality of deoxyribonucleotide triphosphates each comprising a modified base having a photocleavable carbon chain moiety, which when present, enables single incorporation.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 054,766, filed July 21, 2020, the contents of which are incorporated herein by reference in their entirety.

[0003] Reference to sequence list

[0004] This application is submitted together with an electronic sequence list. The sequence list is provided as a file created on July 6, 2021, entitled “Sequences_Listing_47CX-311972-US”, which is 1 kilobyte in size. The full text of the electronic sequence list is incorporated herein by reference. Background Technology Technical Field

[0006] This disclosure relates in its entirety to the field of nucleic acid synthesis, which, for example, uses base-modified nucleotides for TdT-based nucleic acid synthesis. Background Technology

[0008] Traditionally, a four-step chemical method based on phosphoramide chemistry is used to synthesize deoxyribonucleic acid (DNA), allowing the synthesis of DNA strands of up to 250 to 300 base pairs. Enzymatic gene synthesis is an alternative for achieving DNA sequence synthesis. Summary of the Invention

[0009] This document discloses embodiments of methods for nucleic acid synthesis. In some embodiments, the method includes (a1) providing a nucleic acid, a first nucleoside triphosphate, and a first terminal deoxynucleotidyl transferase (TdT), the first nucleoside triphosphate comprising a modified base having at least The method may include: (b1) contacting (i) a nucleic acid and (ii) a first nucleoside triphosphate with a first TdT to generate a first modified nucleic acid, the first modified nucleic acid comprising a nucleic acid incorporating a first nucleotide, the first nucleotide containing a modified base from the first nucleoside triphosphate. The method may include (c1) photolyzing the modified base of the first nucleotide in the photolytic carbon chain portion of the first modified nucleic acid to remove the photolytic carbon chain portion from the first modified nucleic acid.

[0010] In some embodiments, the method further includes: (a2) providing a second nucleoside triphosphate and a second TdT, the second nucleoside triphosphate comprising a modified base, the modified base comprising having at least The method may include: (b2) contacting (i) a first modified nucleic acid in which the photolytic carbon chain portion of the modified base of the first nucleotide is removed and (ii) a second nucleoside triphosphate with a second TdT to generate a second modified nucleic acid, the second modified nucleic acid comprising a first modified nucleic acid incorporating a second nucleotide containing a modified base from the second nucleoside triphosphate. The method may include (c2) photolyzing the modified base of the second nucleotide in the photolytic carbon chain portion of the second modified nucleic acid to remove the photolytic carbon chain portion from the second modified nucleic acid.

[0011] The disclosure herein includes implementation schemes for methods of nucleic acid synthesis. In some implementation schemes, the method includes: (a) providing a nucleic acid and a plurality of nucleoside triphosphates, each of the plurality of nucleoside triphosphates comprising a modified base, the modified base comprising having at least The method may include (b1) contacting (i) a nucleic acid and (ii) a first nucleoside triphosphate of the plurality of nucleoside triphosphates with a first terminal deoxynucleotidyl transferase (TdT) to generate a first modified nucleic acid, the first modified nucleic acid comprising a nucleic acid incorporating a first nucleotide, the first nucleotide containing a modified base from the first nucleoside triphosphate. The method may include (c1) photolyzing the modified base of the first nucleotide in the photolytic carbon chain portion of the first modified nucleic acid to remove the photolytic carbon chain portion from the first modified nucleic acid. The method may include (b2) contacting (i) the first modified nucleic acid in which the photolytic carbon chain portion of the modified base of the first nucleotide is removed and (ii) a second nucleoside triphosphate of the plurality of nucleoside triphosphates with a second TdT to generate a second modified nucleic acid, the second modified nucleic acid comprising the first modified nucleic acid incorporating a second nucleotide, the second nucleotide containing a modified base from the second nucleoside triphosphate. The method may include (c2) photolyzing the modified base of the second nucleotide in the photolyzable carbon chain portion of the second modified nucleic acid to remove the photolyzable carbon chain portion from the second modified nucleic acid.

[0012] In some embodiments, the nucleic acid concentration is at least 10 nM. In some embodiments, the nucleic acid comprises single-stranded (ss) nucleic acid. In some embodiments, the nucleic acid comprises deoxyribonucleic acid (DNA). In some embodiments, the nucleic acid comprises at least one ribonucleotide. In some embodiments, the nucleic acid is ligated to a solid support. In some embodiments, the solid support comprises a flow cell surface. In some embodiments, the method further includes separating the modified nucleic acid from the solid support.

[0013] In some embodiments, the concentration of the first nucleoside triphosphate and / or the second nucleoside triphosphate is at least 0.1 μM. In some embodiments, the modified bases include modified cytosine, modified uracil, modified thymine, modified adenine, or modified guanine. In some embodiments, the modified bases include propargylamino, aminoallyl, propargylhydroxy, or combinations thereof.

[0014] In some embodiments, the photolyzable carbon chain portion comprises a saturated or unsaturated, substituted or unsubstituted, straight or branched carbon chain. In some embodiments, the carbon chain has at least The length of the photolytic carbon chain portion. In some embodiments, the photolytic carbon chain portion includes at least 54 carbon atoms, oxygen atoms, nitrogen atoms, and / or sulfur atoms in the main chain of the carbon chain. In some embodiments, the photolytic carbon chain portion includes multiple repeating units. In some embodiments, the multiple repeating units include the same repeating unit. In some embodiments, one of the repeating units includes at least three carbon atoms, oxygen atoms, nitrogen atoms, and / or sulfur atoms in the main chain of the repeating unit. In some embodiments, the multiple repeating units include polyethylene glycol (PEG). In some embodiments, the repeating units in the multiple repeating units do not include aromatic groups. In some embodiments, the repeating units in the multiple repeating units include aromatic groups. In some embodiments, the number of multiple repeating units is at least 18.

[0015] In some embodiments, the photolyzable carbon chain portion includes a photolyzable portion selected from the group consisting of: carbonyl group, arylcarbonylmethyl group, benzoylmethyl group, o-alkylbenzoylmethyl group, p-hydroxybenzoylmethyl group, diphenylethanol ketone group, benzyl group, nitroaryl group, nitrobenzyl group, o-nitrobenzyl group, o-nitro-2-phenylethyloxycarbonyl group, o-nitroaniline, coumarin-4-ylmethyl group, arylmethyl group, coumarin group, o-hydroxyarylmethyl group, metal-containing group, neopentylyl group, ester of carboxylic acid, arylsulfonyl group, ketone group, negative carbon atom mediator The following groups are permitted: silyl groups, silyl groups, 2-hydroxycinnamyl groups, α-ketoamide groups, α,β-unsaturated aniline, methyl (phenyl)thiocarbamate groups, S,S-sulfur dioxide chromone groups, 2-pyrrolidine-1,4-benzoquinone groups, triazine groups, arylmethylene imino groups, xanthracene groups, pyronin groups, 7-hydroxy-1,1-dimethylnaphthone groups, carboxylic acid groups, phosphate groups, phosphite groups, sulfate groups, acid groups, alcohol groups, thiol groups, N-oxide groups, phenolic groups, amine groups, derivatives of any of the above substances, or combinations thereof.

[0016] In some embodiments, the concentration of the first TdT and / or the second TdT is at least 10 nM. In some embodiments, the first TdT and / or the second TdT comprises recombinant TdT. In some embodiments, the first TdT and the second TdT are identical. In some embodiments, the first TdT and the second TdT comprise the same molecule of TdT. In some embodiments, the first TdT and the second TdT comprise different molecules of TdT. In some embodiments, the first TdT and the second TdT are different TdTs. In some embodiments, the method further includes: removing the first TdT after step (b1) and before step (c1); and removing the second TdT after step (b2) and before step (c2). In some embodiments, the first TdT is attached to the first bead, such that removing the first TdT comprises magnetic removal of the first TdT after step (b1) and before step (c1), and the second TdT is attached to the second bead, and removing the second TdT comprises magnetic removal of the second TdT after step (b2) and before step (c2). In some embodiments, the first magnetic bead and the second magnetic bead are identical. In some embodiments, the method further includes: deactivating the first TdT after step (b1) and before step (c1); and deactivating the second TdT after step (b2) and before step (c2). In some embodiments, deactivating the first TdT includes thermal deactivation of the first TdT, and deactivating the second TdT includes thermal deactivation of the second TdT.

[0017] In some embodiments, the contact in step (b1) is performed for approximately 5 minutes to approximately 20 minutes. The contact in step (b2) can be performed for approximately 5 minutes to approximately 20 minutes. In some embodiments, the contact in step (b1) is performed at approximately 16°C to approximately 58°C. The contact step (b2) can be performed at approximately 16°C to approximately 58°C.

[0018] In some embodiments, the first modified nucleic acid in step (b1) comprises at least 95% nucleic acid. The second modified nucleic acid in step (b2) may comprise at least 95% of the first modified nucleic acid. In some embodiments, at least 95% of the first modified nucleic acid in step (b1) comprises a first modified nucleic acid that includes a nucleic acid incorporating a single first nucleotide from a first nucleoside triphosphate. At least 95% of the second modified nucleic acid in step (b2) may comprise a second modified nucleic acid that includes a first modified nucleic acid incorporating a single second nucleotide from a second nucleoside triphosphate.

[0019] In some embodiments, the photolysis in step (c1) is performed using a first radiation. The photolysis in step (c2) can be performed using a second radiation. In some embodiments, the first and / or second radiation has a power of about 5 watts to about 20 watts. In some embodiments, the first and / or second radiation includes ultraviolet (UV) radiation. In some embodiments, the first and / or second radiation has a wavelength of about 300 nm to about 400 nm. In some embodiments, an ultraviolet (UV) lamp with a power of about 10 watts to about 60 watts is used to generate the first and / or second radiation. In some embodiments, the photolysis in step (c1) and / or the photolysis in step (c2) is performed for about 1 minute to about 20 minutes. In some embodiments, the photolysis in steps (c1) and / or (c2) has an efficiency of at least 90%.

[0020] In some embodiments, the contact in step (b1) and the contact in step (b2) are each completed within approximately 7 minutes. In some embodiments, the photolysis in step (c1) and the photolysis in step (c2) are each completed within approximately 1 minute. In some embodiments, the contact in step (b1) and the photolysis in step (c1) are completed within approximately 10 minutes, and the contact in step (b2) and the photolysis in step (c2) are completed within approximately 10 minutes.

[0021] In some implementations, the method further includes using a polymerase to generate a reverse complementary sequence of the modified nucleic acid.

[0022] This document discloses embodiments of methods for nucleic acid synthesis. In some embodiments, the method includes: (a1) providing nucleic acids. The method may include: iteratively, (a2) providing a nucleoside triphosphate from a plurality of nucleoside triphosphates and a terminal deoxynucleotidyl transferase (TdT), the nucleoside triphosphate comprising a modified base, the modified base comprising having at least The method involves: (a) obtaining a photolyzable carbon chain portion of a certain length; (b) contacting (i) the nucleic acid in (a1) of the first iteration or the modified nucleic acid from the immediately preceding iteration, for any iteration other than the first iteration, in (c) with (ii) a nucleoside triphosphate, with TdT to generate a modified nucleic acid comprising a nucleic acid in (a1) of the first iteration or the modified nucleic acid from the immediately preceding iteration, for any iteration other than the first iteration, in (c) with a nucleotide incorporated therein, the nucleotide containing a modified base from the nucleoside triphosphate; and (c) photolyzing the modified base of the nucleotide in the photolyzable carbon chain portion of the modified nucleic acid to remove the photolyzable carbon chain portion from the modified nucleic acid. This method can generate modified nucleic acids comprising a predetermined sequence.

[0023] In some embodiments, at least 95% of the modified nucleic acids generated after multiple iterations comprise a predetermined sequence. In some embodiments, the multiple iterations include at least 200 iterations. In some embodiments, the method includes receiving a predetermined sequence.

[0024] This document discloses embodiments of multiple nucleoside triphosphates for use in the synthesis of terminal deoxynucleotidyl transferases (TdT). Each of the multiple nucleoside triphosphates may contain a modified base having at least The length of the photolyzable carbon chain portion.

[0025] In some embodiments, the modified bases include modified cytosine, modified uracil, modified thymine, modified adenine, or modified guanine. In some embodiments, the modified bases include propargylamino groups, aminoallyl groups, or combinations thereof.

[0026] In some embodiments, the photolyzable carbon chain portion comprises a saturated or unsaturated, substituted or unsubstituted, straight or branched carbon chain. In some embodiments, the carbon chain has at least The length of the photolytic carbon chain portion. In some embodiments, the photolytic carbon chain portion includes at least 54 carbon atoms, oxygen atoms, nitrogen atoms, and / or sulfur atoms in the main chain of the carbon chain. In some embodiments, the photolytic carbon chain portion includes multiple repeating units. In some embodiments, the multiple repeating units include the same repeating unit. In some embodiments, one of the repeating units includes at least three carbon atoms, oxygen atoms, nitrogen atoms, and / or sulfur atoms in the main chain of the repeating unit. In some embodiments, the multiple repeating units include polyethylene glycol (PEG). In some embodiments, the repeating units in the multiple repeating units do not include aromatic groups. In some embodiments, the repeating units in the multiple repeating units include aromatic groups. In some embodiments, the number of multiple repeating units is at least 18.

[0027] In some embodiments, the photolyzable carbon chain portion includes a photolyzable portion selected from the group consisting of: carbonyl group, arylcarbonylmethyl group, benzoylmethyl group, o-alkylbenzoylmethyl group, p-hydroxybenzoylmethyl group, diphenylethanol ketone group, benzyl group, nitroaryl group, nitrobenzyl group, o-nitrobenzyl group, o-nitro-2-phenylethyloxycarbonyl group, o-nitroaniline, coumarin-4-ylmethyl group, arylmethyl group, coumarin group, o-hydroxyarylmethyl group, metal-containing group, neopentylyl group, ester of carboxylic acid, aromatic Sulfonyl groups, ketones, carbon-negative mediated groups, silyl groups, silyl groups, 2-hydroxycinnamyl groups, α-ketoamides, α,β-unsaturated aniline, methyl (phenyl)thiocarbamate, S,S-sulfur chromone, 2-pyrrolidine-1,4-benzoquinone groups, triazine groups, arylmethylene imino groups, xanthracene groups, pyronin groups, 7-hydroxy-1,1-dimethylnaphthone, carboxylic acids, phosphates, phosphites, sulfates, acids, alcohols, thiols, N-oxides, phenols, amines, derivatives of any of the above substances, or combinations thereof.

[0028] In some embodiments, the photolyzable portion can be photolyzed with an efficiency of at least 90% over a period of about 1 minute to about 20 minutes by radiation with a power of about 5 watts to about 20 watts. In some embodiments, the radiation includes ultraviolet (UV) radiation. In some embodiments, the radiation has a wavelength of about 300 nm to about 400 nm.

[0029] Details of one or more specific embodiments of the subject matter described in this specification are set forth in the accompanying drawings and the following description. Other features, aspects, and advantages will become apparent from the specification, drawings, and claims. The summary of this invention and the following detailed description are not intended to limit or restrict the scope of the subject matter. Attached Figure Description

[0030] Figure 1A : The structure of nucleotides 1 to 4. Figure 1B The structure of nucleotides 5 to 8.

[0031] Figure 2A : This shows a modified TBE-urea gel with the incorporation of compounds 1 to 4 via TdT. Figure 2B : A bar graph showing the effect of PEG length on the yield of 22-nt oligonucleotides.

[0032] Figure 3 Selective incorporation of compound 5 via TdT was observed at 7 minutes of incubation, with the incorporation time increasing from 5 minutes to 9 minutes.

[0033] Figure 4Compared to 3'OH-modified nucleotides, TdT can be advantageously incorporated into base-modified nucleotides.

[0034] Figure 5 Two cycles of incorporation and deprotection were conducted to demonstrate the feasibility of using compound 5 for the synthesis of enzyme oligonucleotides.

[0035] Figure 6 : Incorporation of nucleotides 5 to 8 via TdT.

[0036] Figure 7 Study on UV photolysis efficiency.

[0037] Throughout the accompanying drawings, reference numerals may be used repeatedly to indicate correspondences between reference elements. The drawings are provided to illustrate exemplary embodiments described herein and are not intended to limit the scope of this disclosure. Detailed Implementation

[0038] In the following detailed description, reference is made to the accompanying drawings, which form part of the detailed description. In the drawings, like reference numerals generally identify like components unless the context otherwise requires. The exemplary embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter set forth herein. It will be readily understood that, as generally described herein and as illustrated in the drawings, aspects of this disclosure can be arranged, substituted, combined, separated, and designed into a variety of different configurations, all of which are expressly covered herein and form part of this disclosure.

[0039] All patents, published patent applications, other publications and sequences from GenBank, and other databases mentioned herein are incorporated by reference in full with respect to the relevant art.

[0040] Traditionally, a four-step chemical method based on phosphoramide chemistry has been used to synthesize DNA. By controlling depurination, the synthesis of oligonucleotides with lengths of 250 to 300 nucleotides can be achieved. For example, enzyme gene synthesis using terminal deoxynucleotidyl transferase (TdT) is an alternative for achieving DNA sequence synthesis.

[0041] TdT enzymes (also known as misleading polymerases) are unique polymerases because they do not require a template strand for oligonucleotide synthesis. Since TdT can incorporate nucleotides indiscriminately, it can be used to achieve enzyme gene synthesis. However, when using unmodified nucleotides, TdT can incorporate more than 8000 bases in 24 hours. For TdT that can be used for enzyme gene synthesis, it can perform single incorporation each time a specific nucleotide is introduced. This single incorporation allows for the correct sequence of the desired DNA oligonucleotide to be synthesized. If multiple incorporations occur each time a specific nucleotide is introduced, there will be no control in the synthesized sequence.

[0042] There are two possible strategies to achieve controlled single incorporation events using TdT. The 3' hydroxyl (3'-OH) group of the nucleotide can be modified with a reversible capping group, or the nucleobase can be modified with a reversible capping group that prevents more than one incorporation. These reversible cappings can then be removed after the incorporation event to allow for the next incorporation.

[0043] The use of 3' hydroxyl capping groups on nucleotides requires the engineering of native TdT polymerases to accommodate larger 3' cappings in the enzyme's active site. Modification of human TdT at the nucleotide-binding domain can lead to a significant loss of activity and stability. When residues near the nucleotide-binding side are mutated, only 3% to 16% of TdT activity is retained. TdT has evolved to attempt to incorporate 3' capped nucleotides.

[0044] A second strategy to achieve a single incorporation event is to have a capping group at the nucleobase while leaving the 3' hydroxyl position uncapped. Modification at the 3' hydroxyl group directly affects the enzyme's active site. However, modifications at the C5 position of the pyrimidine in the nucleotide or the C7 position of the 7-deazine in the nucleotide extend further away from the enzyme's active site and are more easily tolerated by polymerases. The development of nucleobase modifications that can prevent subsequent incorporation involves nucleotide engineering to optimize the size and properties of the capping group, such as lipophilicity. Furthermore, TdT should be used to efficiently incorporate the modified nucleotide and prevent subsequent incorporation after its incorporation.

[0045] This document discloses embodiments of methods for nucleic acid synthesis. In some embodiments, the method includes (a1) providing a nucleic acid, a first nucleoside triphosphate, and a first terminal deoxynucleotidyl transferase (TdT), the first nucleoside triphosphate comprising a modified base having at least The method may include: (b1) contacting (i) a nucleic acid and (ii) a first nucleoside triphosphate with a first TdT to generate a first modified nucleic acid, the first modified nucleic acid comprising a nucleic acid incorporating a first nucleotide, the first nucleotide containing a modified base from the first nucleoside triphosphate. The method may include (c1) photolyzing the modified base of the first nucleotide in the photolytic carbon chain portion of the first modified nucleic acid to remove the photolytic carbon chain portion from the first modified nucleic acid.

[0046] The disclosure herein includes implementation schemes for methods of nucleic acid synthesis. In some implementation schemes, the method includes: (a) providing a nucleic acid and a plurality of nucleoside triphosphates, each of the plurality of nucleoside triphosphates comprising a modified base, the modified base comprising having at least The method may include (b1) contacting (i) a nucleic acid and (ii) a first nucleoside triphosphate of the plurality of nucleoside triphosphates with a first terminal deoxynucleotidyl transferase (TdT) to generate a first modified nucleic acid, the first modified nucleic acid comprising a nucleic acid incorporating a first nucleotide, the first nucleotide containing a modified base from the first nucleoside triphosphate. The method may include (c1) photolyzing the modified base of the first nucleotide in the photolytic carbon chain portion of the first modified nucleic acid to remove the photolytic carbon chain portion from the first modified nucleic acid. The method may include (b2) contacting (i) the first modified nucleic acid in which the photolytic carbon chain portion of the modified base of the first nucleotide is removed and (ii) a second nucleoside triphosphate of the plurality of nucleoside triphosphates with a second TdT to generate a second modified nucleic acid, the second modified nucleic acid comprising the first modified nucleic acid incorporating a second nucleotide, the second nucleotide containing a modified base from the second nucleoside triphosphate. The method may include (c2) photolyzing the modified base of the second nucleotide in the photolyzable carbon chain portion of the second modified nucleic acid to remove the photolyzable carbon chain portion from the second modified nucleic acid.

[0047] This document discloses embodiments of methods for nucleic acid synthesis. In some embodiments, the method includes: (a1) providing nucleic acids. The method may include: iteratively, (a2) providing a nucleoside triphosphate from a plurality of nucleoside triphosphates and a terminal deoxynucleotidyl transferase (TdT), the nucleoside triphosphate comprising a modified base, the modified base comprising having at least The method involves: (a) obtaining a photolyzable carbon chain portion of a certain length; (b) contacting (i) the nucleic acid in (a1) of the first iteration or the modified nucleic acid from the immediately preceding iteration, for any iteration other than the first iteration, in (c) with (ii) a nucleoside triphosphate, with TdT to generate a modified nucleic acid comprising a nucleic acid in (a1) of the first iteration or the modified nucleic acid from the immediately preceding iteration, for any iteration other than the first iteration, in (c) with a nucleotide incorporated therein, the nucleotide containing a modified base from the nucleoside triphosphate; and (c) photolyzing the modified base of the nucleotide in the photolyzable carbon chain portion of the modified nucleic acid to remove the photolyzable carbon chain portion from the modified nucleic acid. This method can generate modified nucleic acids comprising a predetermined sequence.

[0048] This document discloses embodiments of multiple nucleoside triphosphates for use in the synthesis of terminal deoxynucleotidyl transferases (TdT). Each of the multiple nucleoside triphosphates may contain a modified base having at least The length of the photolyzable carbon chain portion.

[0049] Enzyme nucleic acid synthesis

[0050] A four-step chemical approach based on phosphoramide chemistry is used to synthesize deoxyribonucleic acid (DNA), allowing the synthesis of DNA strands of up to 250 to 300 base pairs. Enzymatic gene synthesis is an alternative for achieving the synthesis of DNA sequences, such as long DNA sequences. Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that can be used for this type of enzyme-based gene synthesis. To achieve single nucleotide incorporation when using TdT, reversible capping groups can be present at the 3' hydroxyl position or at the nucleobase of the nucleotide.

[0051] Two incorporation reactions and photolysis reaction

[0052] This document discloses methods for nucleic acid synthesis. In some embodiments, the method for nucleic acid synthesis includes: (a) providing a nucleic acid and a plurality of nucleoside triphosphates. Each of the plurality of nucleoside triphosphates may contain a modified base. The modified base may include having at least The length of the photolyzable carbon chain portion.

[0053] The nth incorporation reaction and photolysis reactionThe method may include (b1) contacting (i) a nucleic acid and (ii) a first nucleoside triphosphate of the plurality of nucleoside triphosphates with a first terminal deoxynucleotidyl transferase (TdT) to generate a first modified nucleic acid. The first modified nucleic acid may include a nucleic acid incorporating a first nucleotide, the first nucleotide containing a modified base from the first nucleoside triphosphate. The method may include (c1) photolyzing the modified base of the first nucleotide in the photolyzable carbon chain portion of the first modified nucleic acid to remove the photolyzable carbon chain portion from the first modified nucleic acid.

[0054] The (n+1)th incorporation reaction and photolysis reaction The method may include (b2) contacting (i) a first modified nucleic acid in which the photolytic carbon chain portion of the modified base of the first nucleotide is removed, and (ii) contacting a second nucleoside triphosphate of the plurality of nucleoside triphosphates with a second TdT to generate a second modified nucleic acid. The second modified nucleic acid may include a first modified nucleic acid incorporating a second nucleotide comprising a modified base from the second nucleoside triphosphate. The method may include (c2) photolyzing the photolytic carbon chain portion of the modified base of the second nucleotide in the second modified nucleic acid to remove the photolytic carbon chain portion from the second modified nucleic acid.

[0055] Multiple incorporation reactions and photolysis reactions

[0056] In some embodiments, the method of nucleic acid synthesis includes (a1) providing nucleic acids. This method may include: iteratively, (a2) providing a nucleoside triphosphate from a plurality of nucleoside triphosphates and a terminal deoxynucleotidyl transferase (TdT), the nucleoside triphosphate comprising a modified base having at least The method may include (b) contacting (i) a nucleic acid in (a1) of a first iteration or a modified nucleic acid from an immediately preceding iteration, for any iteration other than the first iteration, and (ii) a nucleoside triphosphate with TdT to generate a modified nucleic acid comprising a nucleotide incorporated in (a1) of the nucleic acid in the first iteration or a modified nucleic acid from an immediately preceding iteration, for any iteration other than the first iteration, the nucleotide containing a modified base from the nucleoside triphosphate. The method may include (c) photolyzing the modified base of the nucleotide in the photolytic carbon chain portion of the modified nucleic acid to remove the photolytic carbon chain portion from the modified nucleic acid. The method may generate a modified nucleic acid comprising a predetermined sequence. The method may include receiving a predetermined sequence.

[0057] The varying percentages of modified nucleic acids generated after multiple iterations may include predetermined sequences. The number of iterations may vary across different implementations. In some implementations, the number of iterations includes, includes about, includes at least, includes at least about, includes at most, or includes at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 iterations. The iterations can be 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 7000, 8000, 9000, 10000, or a value or range between any two of these values. For example, this multiple iteration includes at least 200 iterations.

[0058] Remove TdT

[0059] In some embodiments, the method includes removing TdT used after the doping reaction or contact step and before the photolysis step. For example, the method includes removing a first TdT after step (b1) and before step (c1). As another example, the method may include removing a second TdT after step (b2) and before step (c2). In some embodiments, the first TdT is attached to a first magnetic bead or particle. Removing the first TdT may include magnetically removing the first TdT after step (b1) and before step (c1). The second TdT may be attached to a second magnetic bead or particle. Removing the second TdT may include magnetically removing the second TdT after step (b2) and before step (c2). In some embodiments, the first magnetic bead and the second magnetic bead are identical. In some embodiments, the first TdT and the second TdT are identical. In some embodiments, the first TdT and the second TdT comprise the same molecules of TdT. In some embodiments, the first TdT and the second TdT comprise different molecules of TdT.

[0060] Inactivate TdT

[0061] In some embodiments, the method includes deactivating TdT used after the incorporation reaction or contact step and before the photolysis step. For example, the method may include deactivating a first TdT after step (b1) and before step (c1). The method may include deactivating a second TdT after step (b2) and before step (c2). In some embodiments, deactivating the first TdT includes thermally deactivating the first TdT, and deactivating the second TdT may include thermally deactivating the second TdT.

[0062] Nucleic acid

[0063] The concentration of nucleic acid can vary in different implementation schemes. In some implementation schemes, the concentration of nucleic acid is specified as: about, at least, at least about, at most, or at most about 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM. 90 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, or values ​​or ranges between any two of these values. For example, the concentration of nucleic acid is at least 10 nM.

[0064] In some embodiments, the nucleic acid comprises a single-stranded (ss) nucleic acid. In some embodiments, the nucleic acid comprises a double-stranded (ds) nucleic acid having a 3' overhang. In some embodiments, the nucleic acid comprises a double-stranded nucleic acid having a 3' recessed end. In some embodiments, the nucleic acid comprises deoxyribonucleic acid (DNA). In some embodiments, the nucleic acid comprises at least one ribonucleotide.

[0065] In some embodiments, nucleic acids are ligated to a solid support. The nucleic acids can be covalently or non-covalently ligated to the solid support. The nucleic acids can be directly or indirectly ligated to the solid support. The nucleic acids can be conjugated to the solid support. The solid support can be or comprise beads or particles. The solid support can be non-magnetic, magnetic, or paramagnetic. The solid support can be or comprise a flow cell surface. In some embodiments, the method includes separating the modified nucleic acid from the solid support.

[0066] Nucleoside triphosphate

[0067] In different embodiments, the concentration of the nucleoside triphosphates (e.g., first nucleoside triphosphate and second nucleoside triphosphate) disclosed herein may vary. In some embodiments, the concentration of the nucleoside triphosphates is, is about, is at least, is at least about, is at most or is at most about 0.001 μM, 0.002 μM, 0.003 μM, 0.004 μM, 0.005 μM, 0.006 μM, 0.007 μM, 0.008 μM, 0.009 μM, 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, or a value or range between any two of these values. For example, the concentration of nucleoside triphosphate is at least 0.1 μM.

[0068] base

[0069] In some embodiments, the modified bases include modified cytosine (c), modified uracil (U), modified thymine (T), modified adenine (a), or modified guanine (G). In some embodiments, the modified bases include propargylamino, aminoallyl, propargylhydroxy, or combinations thereof. For example, the modified bases may be propargylaminocytosine, propargylaminouracil, propargylaminothymine, propargylaminoadenine, or propargylaminoguanine. For example, the modified bases may be aminoallylcytosine, aminoallyluracil, aminoallylthymine, aminoallyladenine, or aminoallylguanine. For example, the modified bases may be propargylhydroxycytosine, propargylhydroxyuracil, propargylhydroxythymine, propargylhydroxyadenine, or propargylhydroxyguanine.

[0070] Photolytic carbon chain

[0071] In some embodiments, the photolyzable carbon chain portion comprises a saturated or unsaturated, substituted or unsubstituted, straight or branched carbon chain. The length of the photolyzable carbon chain may vary in different embodiments. In some embodiments, the photolyzable carbon chain has a length that is, is about, is at least, is at least about, is at most, or is at most about Or a value or range between any two of these values. For example, a carbon chain has at least The length.

[0072] In various embodiments, the photolyzable carbon chain portion may include 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 combinations thereof. In some embodiments, the photolyzable carbon chain portion includes, includes about, includes at least, includes at least about, includes at most or includes at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 2 8, 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 1, 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 The photodegradable carbon chain portion comprises 1, 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, of carbon, oxygen, nitrogen, sulfur, or combinations thereof. For example, the photodegradable carbon chain portion comprises at least 54 carbon, oxygen, nitrogen, and / or sulfur atoms. In various embodiments, the photodegradable carbon chain portion may include 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 combinations thereof, within the main chain of the photodegradable carbon chain portion.In some embodiments, the photolyzable carbon chain moiety includes, includes about, includes at least, includes at least about, includes at most or includes at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 of the carbon chain moiety in the main chain of the carbon chain moiety. 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 1, 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, 1 00, 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, of carbon, oxygen, nitrogen, sulfur, or combinations thereof. For example, the photolytic carbon chain portion comprises at least 54 carbon, oxygen, nitrogen, and / or sulfur atoms in the main chain of the photolytic carbon chain.

[0073] In some embodiments, the photodegradable carbon chain portion comprises a polymer, such as a homopolymer or heteropolymer. In some embodiments, the photodegradable carbon chain portion comprises a plurality of repeating units. In some embodiments, the plurality of repeating units comprises the same repeating unit. In some embodiments, the plurality of repeating units comprises two or more different repeating units. The plurality of repeating units may comprise polyethers, such as paraformaldehyde, polyethylene glycol (PEG), polypropylene glycol (PPG), polyalkylene glycol (PAG), polytetramethylene glycol (PTMG), or combinations thereof. For example, the plurality of repeating units may comprise PEG. 18 PEG 23 PEG 24Or combinations thereof. The plurality of repeating units may include polyalkylene compounds, such as polyethylene, polypropylene, polybutene, or combinations thereof. In some embodiments, the repeating units in the plurality of repeating units do not include aromatic groups. In some embodiments, the repeating units in the plurality of repeating units include one or more aromatic groups.

[0074] The repeating unit in the plurality of repeating units may include 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 combinations thereof. For example, the repeating unit in the plurality of repeating units may include ethylene oxide, which comprises two carbon atoms and one oxygen atom. In some embodiments, one or each of the plurality of repeating units includes, includes about, includes at least, includes at least about, includes at most or includes at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 carbon atoms, oxygen atoms, nitrogen atoms, sulfur atoms, or combinations thereof. For example, one or each of the plurality of repeating units comprises a saturated or unsaturated, substituted or unsubstituted, straight or branched carbon chain. For example, one or more repeating units comprise a C1 alkyl group, a C2 alkyl group, a C3 alkyl group, a C4 alkyl group, a C5 alkyl group, or a C6 alkyl group.

[0075] The repeating units in the plurality of repeating units may include 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 combinations thereof, in the main chain of the repeating units. For example, the repeating units in the plurality of repeating units may include ethylene oxide in the main chain of the repeating units, which comprises two carbon atoms and one oxygen atom. In some implementations, one or each of the plurality of repeating units includes, includes about, includes at least, includes at least about, includes at most or includes at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46 repeating units in the main chain of repeating units. 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 carbon atoms, oxygen atoms, nitrogen atoms, sulfur atoms, or combinations thereof. For example, one or each of the plurality of repeating units includes a saturated or unsaturated, substituted or unsubstituted carbon chain in the main chain of the repeating unit. For example, one or each of the plurality of repeating units includes a saturated or unsaturated, substituted or unsubstituted alkyl group in the main chain of the repeating unit. For example, one or each of the plurality of repeating units includes a methyl group, ethyl group, propyl group, butyl group, or hexyl group in the main chain of the repeating unit. For example, one or each of the plurality of repeating units includes a methoxy group, ethoxy group, propoxy group, butoxy group, or hexoxy group in the main chain of the repeating unit.

[0076] In different implementations, the number of repeating units can vary. In some implementations, the number of repeating units is: about, at least, at least about, at most or at most about 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 1, 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, or a number or range between any two of these values. For example, the number of repeating units is at least 18, 23, or 24. For example, the repeating units may include PEG. 18 PEG 23 PEG 24 Or a combination thereof.

[0077] Photolytically degradable portion

[0078] In some embodiments, the photolyzable carbon chain portion includes a photolyzable portion selected from the group consisting of: carbonyl group, arylcarbonylmethyl group, benzoylmethyl group, o-alkylbenzoylmethyl group, p-hydroxybenzoylmethyl group, diphenylethanol ketone group, benzyl group, nitroaryl group, nitrobenzyl group, o-nitrobenzyl group, o-nitro-2-phenylethyloxycarbonyl group, o-nitroaniline, coumarin-4-ylmethyl group, arylmethyl group, coumarin group, o-hydroxyarylmethyl group, metal-containing group, neopentylyl group, ester of carboxylic acid, arylsulfonyl group, ketone group, negative carbon atom mediator The following groups are permitted: silyl groups, silyl groups, 2-hydroxycinnamyl groups, α-ketoamide groups, α,β-unsaturated aniline, methyl (phenyl)thiocarbamate groups, S,S-sulfur dioxide chromone groups, 2-pyrrolidine-1,4-benzoquinone groups, triazine groups, arylmethylene imino groups, xanthracene groups, pyronin groups, 7-hydroxy-1,1-dimethylnaphthone groups, carboxylic acid groups, phosphate groups, phosphite groups, sulfate groups, acid groups, alcohol groups, thiol groups, N-oxide groups, phenolic groups, amine groups, derivatives of any of the above substances, or combinations thereof.

[0079] TdT

[0080] In different embodiments, the concentration of TdT (e.g., first TdT and second TdT) disclosed herein may vary. In some embodiments, the concentration of TdT is, is about, is at least, is at least about, is at most or is at most about 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, or 80 nM. 90 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, or values ​​or ranges between any two of these values. For example, the concentration of TdT is at least 10 nM.

[0081] In some embodiments, TdT (e.g., first TdT and second TdT) comprises recombinant TdT. In some embodiments, first TdT and second TdT are identical. In some embodiments, first TdT and second TdT comprise the same molecules of TdT. In some embodiments, first TdT and second TdT comprise different molecules of TdT. The same or different molecules of TdT can be used in multiple incorporation reactions. For example, molecules of TdT can be attached to one or more magnetic beads or particles. During the nth contact step, TdT can be introduced into the nth incorporation reaction by introducing magnetic beads or particles. After the nth incorporation reaction or contact step and before the nth photolysis reaction or step, the method may include removing molecules of TdT from the nth incorporation reaction by magnetically removing the magnetic beads or particles from the nth incorporation reaction. During the (n+1)th contact step, TdT can be introduced into the (n+1)th contact reaction by introducing the same magnetic beads or particles used in the nth doping reaction or contact step. In some embodiments, the first TdT and the second TdT comprise different TdTs.

[0082] Incorporation reaction

[0083] In different embodiments, the doping reaction or contacting steps of this disclosure may occur or be performed at different doping reaction temperatures (e.g., the nth doping reaction or contacting step in (b1) and the (n+1)th doping reaction or contacting step in (b2)). In some embodiments, the doping reaction temperature is, about, at least, at least about, at most, or at most about 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 4... 3°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, or a value or range between any two of these values. For example, the doping reaction or contact step is performed at about 16°C to about 58°C.

[0084] In different embodiments, the efficiency of the incorporation reaction or contact step of this disclosure (e.g., the nth incorporation reaction or contact step, or the (n+1)th incorporation reaction or contact step) may vary. In some embodiments, the efficiency of the incorporation reaction or contact step of this disclosure is, about, at least, at least about, at most, or at most about 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%, or 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%, 99.9%, 99.99%, 100%, or a value or range between any two of these values. For example, the first modified nucleic acid in step (b1) comprises at least 95% nucleic acid incorporating a first nucleoside triphosphate. For example, the second modified nucleic acid in step (b2) comprises at least 95% of the first modified nucleic acid, into which a second nucleoside triphosphate is incorporated. For instance, at least 95% of the first modified nucleic acid in step (b1) comprises a first modified nucleic acid, which includes a nucleic acid incorporating a single first nucleotide from a first nucleoside triphosphate. At least 95% of the second modified nucleic acid in step (b2) may include a second modified nucleic acid, which comprises a first modified nucleic acid incorporating a single second nucleotide from a second nucleoside triphosphate.

[0085] In some implementations, the percentage of modified nucleic acids generated after multiple iterations is: about, at least, at least about, at most or at most about 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%, 5 3%, 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%, 99.9%, 99.99%, 100%, or a value or range between any two of these values. For example, at least 95% of the modified nucleic acids generated after multiple iterations comprise a predetermined sequence.

[0086] Photolysis reaction

[0087] The photolysis reaction or photolysis step of this disclosure can be performed using radiation (e.g., the nth photolysis reaction or photolysis in step (c1), and the (n+1)th photolysis reaction or photolysis in step (c2)). For example, the nth photolysis reaction or photolysis in step (c1) can be performed using first radiation. The (n+1)th photolysis reaction or photolysis in step (c2) can be performed using second radiation. The first radiation and the second radiation can be the same. The first radiation and the second radiation can be different. In different embodiments, the radiation for the photolysis reaction or photolysis step can be different. In some embodiments, the radiation from the photolysis reaction or photolysis step has power, which is, about, at least, at least about, at most, or at most about 1 watt, 2 watts, 3 watts, 4 watts, 5 watts, 6 watts, 7 watts, 8 watts, 9 watts, 10 watts, 11 watts, 12 watts, 13 watts, 14 watts, 15 watts, 16 watts, 17 watts, 18 watts, 19 watts, or 20 watts. 21 watts, 22 watts, 23 watts, 24 watts, 25 watts, 26 watts, 27 watts, 28 watts, 29 watts, 30 watts, 31 watts, 32 watts, 33 watts, 34 watts, 35 watts, 36 watts, 37 watts, 38 watts, 39 watts, 40 watts, 41 watts, 42 watts, 43 watts, 44 watts, 45 watts, 46 watts, 47 watts, 48 ​​watts 49 watts, 50 watts, 51 watts, 52 watts, 53 watts, 54 watts, 55 watts, 56 watts, 57 watts, 58 watts, 59 watts, 60 watts, 61 watts, 62 watts, 63 watts, 64 watts, 65 watts, 66 watts, 67 watts, 68 watts, 69 watts, 70 watts, 71 watts, 72 watts, 73 watts, 74 watts, 75 watts, 76 watts 77 watts, 78 watts, 79 watts, 80 watts, 81 watts, 82 watts, 83 watts, 84 watts, 85 watts, 86 watts, 87 watts, 88 watts, 89 watts, 90 watts, 91 watts, 92 watts, 93 watts, 94 watts, 95 watts, 96 watts, 97 watts, 98 watts, 99 watts, 100 watts, or values ​​or ranges between any two of these values. For example, the first radiation used in the nth photolysis reaction or photolysis in step (c1) and the second radiation used in the (n+1)th photolysis reaction or photolysis in step (c2) have a power of about 5 watts to about 20 watts.

[0088] In some embodiments, the radiation used in the photolysis reaction or photolysis step includes ultraviolet (UV) radiation. In different embodiments, the radiation may have different wavelengths. In some embodiments, the radiation has a wavelength of, is about, is at least, is at least about, is at most, or is at most about 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 2 0.5nm, 210nm, 215nm, 220nm, 225nm, 230nm, 235nm, 240nm, 245nm, 250nm, 255nm, 260nm, 265nm, 270nm, 275nm, 280nm, 285nm, 290nm, 295nm, 300nm, 305nm, 310nm, 315nm, 320nm, 325nm, 330nm, 335nm, 340nm, 345nm, 350nm, 355nm, 360nm, 365nm, 370nm, 375nm, 380nm, 385nm, 390nm, 395nm, 400nm, or a value or range between any two of these values. For example, the first radiation used in the nth photolysis reaction or photolysis in step (c1) and the second radiation used in the (n+1)th photolysis reaction or photolysis in step (c2) have wavelengths of about 300 nm to about 400 nm.

[0089] Light (such as ultraviolet (UV) lamps) can be used to generate radiation used in the photolysis reaction or photolysis step. In different embodiments, the power of the lamp used to generate the radiation can vary. In some embodiments, the lamp used to generate the radiation has a power of, about, at least, at least about, at most, or at most about 1 watt, 2 watts, 3 watts, 4 watts, 5 watts, 6 watts, 7 watts, 8 watts, 9 watts, 10 watts, 11 watts, 12 watts, 13 watts, 14 watts, 15 watts, 16 watts, 17 watts, 18 watts, 19 watts, 20 watts, 2 1 watt, 22 watts, 23 watts, 24 watts, 25 watts, 26 watts, 27 watts, 28 watts, 29 watts, 30 watts, 31 watts, 32 watts, 33 watts, 34 watts, 35 watts, 36 watts, 37 watts, 38 watts, 39 watts, 40 watts, 41 watts, 42 watts, 43 watts, 44 watts, 45 watts, 46 watts, 47 watts, 48 ​​watts, 4 9 watts, 50 watts, 51 watts, 52 watts, 53 watts, 54 watts, 55 watts, 56 watts, 57 watts, 58 watts, 59 watts, 60 watts, 61 watts, 62 watts, 63 watts, 64 watts, 65 watts, 66 watts, 67 watts, 68 watts, 69 watts, 70 watts, 71 watts, 72 watts, 73 watts, 74 watts, 75 watts, 76 watts, 7 7 watts, 78 watts, 79 watts, 80 watts, 81 watts, 82 watts, 83 watts, 84 watts, 85 watts, 86 watts, 87 watts, 88 watts, 89 watts, 90 watts, 91 watts, 92 watts, 93 watts, 94 watts, 95 watts, 96 watts, 97 watts, 98 watts, 99 watts, 100 watts, or a value or range between any two of these values. For example, a lamp with a power of about 10 watts to about 60 watts is used to generate the first radiation used in the nth photolysis reaction or photolysis in step (c1) and the second radiation used in the (n+1)th photolysis reaction or photolysis in step (c2).

[0090] In different embodiments, the photolysis reaction or photolysis step can have different photolysis efficiencies. In some embodiments, the photolysis reaction or photolysis step has a photolysis efficiency of, about, at least, at least about, at most, or at most about 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%, or 50%. 3%, 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%, 99.9%, 99.99%, 100%, or a value or range between any two of these values. For example, the nth photolysis reaction or photolysis in step (c1) and the (n+1)th photolysis reaction or photolysis in step (c2) each have a photolysis efficiency of at least 90%.

[0091] In different embodiments, the photolysis reaction or photolysis step of this disclosure can occur or be performed at different photolysis reaction temperatures (e.g., the nth photolysis reaction or photolysis step in (x1) and the (n+1)th photolysis reaction or photolysis step in (c2)). In some embodiments, the photolysis reaction temperature is, about, at least, at least about, at most, or at most about 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 4... 3℃, 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℃, or a value or range between any two of these values. For example, the nth photolysis reaction or photolysis in step (c1) and the (n+1)th photolysis reaction or photolysis in step (c2) are each performed at approximately 16℃ to approximately 58℃.

[0092] reaction time

[0093] In different embodiments, the doping reaction or contacting steps of this disclosure may be performed or completed within different doping reaction times (e.g., the nth doping reaction or contacting step in (b1) and the (n+1)th doping reaction or contacting step in (b2)). In some embodiments, the doping reaction time is, about, at least, at least about, at most, or at most about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, or a value or range between any two of these values. For example, the doping reaction or contacting step may be performed or completed within about 5 minutes to about 20 minutes. For example, the incorporation reaction or contact step is carried out or completed in about 7 minutes.

[0094] The photolysis reaction or photolysis step (e.g., photolysis in step (c1) and photolysis in step (c2)) can be performed or completed within different photolysis reaction times. In some embodiments, the photolysis reaction time is, about, at least, at least about, at most, or at most about 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 11 seconds, 12 seconds, 13 seconds, 14 seconds, 15 seconds, 16 seconds, 17 seconds, 18 seconds, 19 seconds, 20 seconds, 21 seconds, 22 seconds, 23 seconds, 24 seconds, 25 seconds, 26 seconds, 27 seconds, 28 seconds, 29 seconds, 30 seconds, 31 seconds, 32 seconds, 33 seconds, 34 seconds, 35 seconds, 36 seconds, 37 seconds, 38 seconds, 39 seconds, 40 seconds, 41 seconds, 42 seconds, 43 seconds, 44 seconds, 45 seconds, or 46 seconds. 47 seconds, 48 ​​seconds, 49 seconds, 50 seconds, 51 seconds, 52 seconds, 53 seconds, 54 seconds, 55 seconds, 56 seconds, 57 seconds, 58 seconds, 59 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, or a value or range between any two of these values. For example, performing or completing a photolysis reaction or photolysis step in approximately 1 minute. Or, performing a photolysis reaction or photolysis step in approximately 1 minute to approximately 20 minutes.

[0095] In different embodiments, the total reaction time of the iterations of (b) the incorporation reaction or contact step and (c) the photolysis reaction or photolysis step may vary. In some embodiments, the total reaction time of the iterations of (b) the incorporation reaction or contact step and (c) the photolysis reaction or photolysis step is, about, at least, at least about, at most or at most about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 2 7 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes, 42 minutes, 43 minutes, 44 minutes, 45 minutes, 46 minutes, 47 minutes, 48 ​​minutes, 49 minutes, 50 minutes, 51 minutes, 52 minutes, 53 minutes, 54 minutes, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, or a value or range between any two of these values. For example, completing the contact in step (b1) and the photolysis in step (c1) within approximately 10 minutes. Similarly, completing the contact in step (b2) and the photolysis in step (c2) within approximately 10 minutes.

[0096] Reverse complementary sequence

[0097] In some embodiments, the method includes using a polymerase to generate an inverse complementary sequence of the modified nucleic acid. The inverse complementary sequence can be generated using a polymerase when the modified nucleic acid is ligated to a solid vector. The inverse complementary sequence can also be generated using a polymerase after the modified nucleic acid has been separated from the solid vector.

[0098] Nucleoside triphosphate

[0099] This document discloses embodiments of multiple nucleoside triphosphates for use in the synthesis of terminal deoxynucleotidyl transferases (TdT). Each of the multiple nucleoside triphosphates may contain a modified base having at least The length of the photolyzable carbon chain portion.

[0100] In some implementations, the photolytic portion can be photolytically irradiated, with the power of the radiation being approximately, at least, at least about, or at most about 1 watt, 2 watts, 3 watts, 4 watts, 5 watts, 6 watts, 7 watts, 8 watts, 9 watts, 10 watts, 11 watts, 12 watts, 13 watts, 14 watts, 15 watts, 16 watts, 17 watts, 18 watts, 19 watts, or 20 watts. 21 watts, 22 watts, 23 watts, 24 watts, 25 watts, 26 watts, 27 watts, 28 watts, 29 watts, 30 watts, 31 watts, 32 watts, 33 watts, 34 watts, 35 watts, 36 watts, 37 watts, 38 watts, 39 watts, 40 watts, 41 watts, 42 watts, 43 watts, 44 watts, 45 watts, 46 watts, 47 watts, 48 ​​watts 49 watts, 50 watts, 51 watts, 52 watts, 53 watts, 54 watts, 55 watts, 56 watts, 57 watts, 58 watts, 59 watts, 60 watts, 61 watts, 62 watts, 63 watts, 64 watts, 65 watts, 66 watts, 67 watts, 68 watts, 69 watts, 70 watts, 71 watts, 72 watts, 73 watts, 74 watts, 75 watts, 76 watts 77 watts, 78 watts, 79 watts, 80 watts, 81 watts, 82 watts, 83 watts, 84 watts, 85 watts, 86 watts, 87 watts, 88 watts, 89 watts, 90 watts, 91 watts, 92 watts, 93 watts, 94 watts, 95 watts, 96 watts, 97 watts, 98 watts, 99 watts, 100 watts, or a value or range between any two of these values.

[0101] In some implementations, the photolyzable portion may be at least, at least about, at most, or at most about 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 11 seconds, 12 seconds, 13 seconds, 14 seconds, 15 seconds, 16 seconds, 17 seconds, 18 seconds, 19 seconds, 20 seconds, 21 seconds, 22 seconds, 23 seconds, 24 seconds, 25 seconds, 26 seconds, 27 seconds, 28 seconds, 29 seconds, 30 seconds, 31 seconds, 32 seconds, 33 seconds, 34 seconds, 35 seconds, 36 seconds, 37 seconds, 38 seconds, 39 seconds, 40 seconds, 41 seconds, 42 seconds, 43 seconds, 44 seconds, 45 seconds, 46 seconds, 47 seconds, or 4... Photolysis by radiation for 8 seconds, 49 seconds, 50 seconds, 51 seconds, 52 seconds, 53 seconds, 54 seconds, 55 seconds, 56 seconds, 57 seconds, 58 seconds, 59 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, or a value or range between any two of these values.

[0102] In some embodiments, the photolyzable portion may be at, about, at least, at least about, at most, or at most about 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, or 4... Temperatures of 4℃, 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℃, or any two of these values, or a range thereof, are determined by radiation photolysis.

[0103] In some implementations, the photolyzable portion can be 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% with efficiencies of approximately 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, respectively. 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%, 99.9%, 99.99%, 100%, or a numerical value or range between any two of these values.

[0104] In some implementations, the photolyzable portion can be photolyzable at wavelengths of approximately, at least, at least about, at most, or at most about 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, etc. The radiative light solution of nm, 210nm, 215nm, 220nm, 225nm, 230nm, 235nm, 240nm, 245nm, 250nm, 255nm, 260nm, 265nm, 270nm, 275nm, 280nm, 285nm, 290nm, 295nm, 300nm, 305nm, 310nm, 315nm, 320nm, 325nm, 330nm, 335nm, 340nm, 345nm, 350nm, 355nm, 360nm, 365nm, 370nm, 375nm, 380nm, 385nm, 390nm, 395nm, 400nm, or any value or range between these values.

[0105] For example, the photolytic portion can be photolytically degraded at an efficiency of at least 90% at 16°C to 58°C over a period of about 1 minute to about 20 minutes by radiation with a power of about 5 watts to about 20 watts. In some embodiments, the radiation includes ultraviolet (UV) radiation. In some embodiments, the radiation has a wavelength of about 300 nm to about 400 nm.

[0106] Example

[0107] Some aspects of the implementation schemes discussed above are further disclosed in detail in the following embodiments, which are not intended to limit the scope of this disclosure in any way.

[0108] Example 1

[0109] Base-modified nucleotides used as substrates for TdT-based enzyme nucleic acid synthesis

[0110] A four-step chemical approach based on phosphoramide chemistry is used to synthesize deoxyribonucleic acid (DNA), allowing the synthesis of DNA strands up to 250 to 300 base pairs. Enzymatic gene synthesis is an alternative for achieving the synthesis of DNA sequences, such as long DNA sequences. Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that can be used for such enzyme-based gene synthesis. To achieve single nucleotide incorporation when using TdT, a reversible capping group can be present at the 3' hydroxyl position or the nucleobase of the nucleotide. This example describes a systematic study of the size of the nucleobase capping group to allow for single incorporation events while leaving the 3' hydroxyl position of the nucleotide unprotected. Polyethylene glycol (PEG) of various lengths was conjugated to the C5 position of the pyrimidine and the C7 position of the 7-deazopurine of the nucleotide. The formation of the desired +1 product in quantitative yield was observed, with PEG24 capped at the nucleobase. The formation of the desired +1 product was demonstrated with all four PEG-modified nucleotides. PEG-modified nucleotides can be used in enzyme DNA synthesis, and the size of the PEG modification can be adjusted to prevent more than one incorporation event.

[0111] Introduction

[0112] Traditionally, a four-step chemical approach based on phosphoramidite chemistry has been used to synthesize DNA. By controlling depurination, oligonucleotides with lengths of 250 to 300 nucleotides can be synthesized. There is a need to synthesize longer DNA fragments in synthetic biology and biotechnology. The ability to synthesize genes is likely important in the synthesis of entire bacterial and yeast genomes and in the discovery of novel proteins. Furthermore, studies have shown that DNA can be an excellent candidate for data storage due to its high 3D density and stability for long-term storage. These developments have driven the demand for the synthesis of longer DNA oligonucleotides. Enzymatic DNA synthesis using terminal deoxynucleotidyl transferases (TdT) is a promising method for synthesizing longer DNA at a lower cost than the phosphoramidite approach (e.g., half the cost).

[0113] TdT enzymes (also known as misleading polymerases) are unique polymerases because they do not require a template strand for oligonucleotide synthesis. Since TdT can incorporate nucleotides indiscriminately, it can be used to achieve enzyme gene synthesis. However, when using unmodified nucleotides, TdT can incorporate more than 8000 bases in 24 hours. For TdT that can be used for enzyme gene synthesis, it can perform single incorporation each time a specific nucleotide is introduced. This single incorporation allows for the correct sequence of the desired DNA oligonucleotide to be synthesized. If multiple incorporations occur each time a specific nucleotide is introduced, there will be no control in the synthesized sequence.

[0114] Two possible strategies exist for achieving controlled single incorporation events using TdT. The 3'-hydroxyl (3'-OH) group of the nucleotide can be modified with a reversible capping group, or the nucleobase can be modified with a reversible capping group that prevents more than one incorporation. These reversible cappings can then be removed after the incorporation event to allow for the next incorporation. Nucleotides with reversibly terminated 3'-OH groups have been extensively studied, particularly in groups developing sequencing-by-synthesis methods. Illumina, Inc. (San Diego, California) developed 3'-O-2'-azidomethyldeoxynucleotide triphosphate as a reversible terminator for sequencing. A palladium-cleavable allyl reversible terminator for sequencing has been developed. An aminooxy capping group has been developed as a reversible terminator for sequencing.

[0115] However, the use of 3' hydroxyl capping groups on nucleotides requires the engineering of native TdT polymerases to accommodate larger 3' cappings in the enzyme's active site. Modifications of human TdT at the nucleotide-binding domain can lead to a significant loss of activity and stability. When residues near the nucleotide-binding side are mutated, only 3% to 16% of TdT activity is retained. TdT has evolved to attempt to incorporate 3' capping nucleotides.

[0116] Applications of nucleobase modification for TdT incorporation

[0117] A second strategy to achieve a single incorporation event is to have a capping group at the nucleotide base while leaving the 3' hydroxyl position uncapped. Modification at the 3' hydroxyl group directly affects the enzyme's active site. However, modifications at the C5 position of the pyrimidine in the nucleotide or the C7 position of the 7-deazine in the nucleotide extend further away from the enzyme's active site and are more easily tolerated by polymerases. The development of nucleotide modifications that can prevent subsequent incorporation involves nucleotide engineering to optimize the size and properties of the capping group, such as lipophilicity. Furthermore, TdT can be used to efficiently incorporate modified nucleotides and prevent subsequent incorporation after their incorporation.

[0118] Several exemplary methods for achieving a single incorporation event are described below. For example, dinucleotides have been used as virtual terminators for sequencing. The nucleotide design involves an uncapped 3'-OH nucleotide, with a second inhibitor nucleotide introduced as a base modification via a disulfide linker. The disulfide linker is then cleaved using a reducing agent such as tris(2-carboxyethyl)phosphine. Another method for achieving a single incorporation event is to attach a diaspartate moiety as a nucleobase capping group via a disulfide linker. Upon cleavage of the capping group, the native nucleobase is obtained without leaving a molecular scar. As another example, TdT mutants have been conjugated to nucleotides where the TdT enzyme serves as a capping group for further incorporation. TdT enzymes include cysteine ​​mutated to alanine or serine, and a single residue near the active site mutated to cysteine. These mutations conjugate maleimide-functionalized nucleotides to the TdT enzyme. TdT-conjugated nucleotides have been observed to exhibit higher incorporation rates compared to unconjugated nucleotides. After incorporation, photolyzable linkers are cleaved using 365 nm UV light. The cleavage step removes the TdT end caps from the growing oligonucleotide chain and may lead to subsequent incorporation events.

[0119] Having N 4Modified nucleotides of -aminocytosine, 4-thiouracil, 2-pyridone, 4-chloro-, and 4-bromo-2-pyridone have been used as nucleobases for TdT incorporation. It is hypothesized that modification of the 3' end of the growing oligonucleotide primer chain enhances TdT-primer affinity. This enhanced TdT-primer affinity can play a role in preventing further incorporation of the incorporating nucleotide. Structural studies of TdT-primer interactions have previously shown that the interaction of the last three nucleobases on the primer oligonucleotide with TdT is required during elongation. Incorporation of benzo[2-(phenylthioyl)ethyl]-2'-deoxy-7-5'-O-deadenyl adenosine triphosphate and 7-[2-(butylthioyl)ethyl]-2'-deoxy-7-5'-O-deadenyl adenosine triphosphate has been shown, and only single incorporation events have been observed.

[0120] This example is a systematic study of the size requirements of the base-capping groups on nucleotides to enable single incorporation using TdT. Nucleotides are conjugated at the nucleobase with polyethylene glycol (PEG) polymers of varying lengths to determine the size requirements for preventing further incorporation events. The 3' hydroxyl position of the nucleotide is left uncapped to minimize its impact on the enzyme's active site. Water-soluble PEG polymer chains are used as capping groups for the systematic study because their length is highly tunable. The PEG groups are attached to the nucleobase via photolyzable nitrobenzyl linkers. These photolyzable nitrobenzyl linkers allow for the removal of the PEG capping groups, enabling subsequent nucleotide incorporation via the TdT enzyme.

[0121] Materials and methods

[0122] Recordings were performed in various solvents using a JEOL ECA 400, Bruker Avance III 400, or Bruker Avance 500 spectrometer. 1 1H NMR spectra (400MHz or 500MHz) [using TMS (for 1 H,δ=0.00), MeOD (for 1 H, δ = 3.31) or D2O (for 1 H, δ = 4.79) was used as an internal standard. Recordings were performed in various solvents on a JEOL ECA 400 spectrometer. 13 CNMR spectrum (100MHz) [using CDCl3 (for 13 C,δ=77.16), MeOD (for 13 C, δ = 49.00) was used as an internal standard. Recordings were performed on a JEOL ECA 400 or Bruker Avance 500 spectrometer. 31P NMR spectra (160 MHz or 202 MHz). The following abbreviations are used to indicate multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, br = broad peak. High-resolution mass spectra were obtained using a Waters Q-Tof Premier mass spectrometer. Chemicals were purchased from Trilink Biotechnology (San Diego, California), Merck Group (Darmstadt, Germany), and Broadpharm (San Diego, California). Anhydrous solvents were purchased from Sigma-Aldrich (St. Louis, Missouri) and Acros Organics (Fair Lawn, New Jersey). TdT was purchased from New England Biolab (Ipswich, Massachusetts). TBE-urea gel (15%) was purchased from Life Technologies. All primers were synthesized by Integrated DNA Technologies (Coralville, Iowa). UV irradiation was performed using a 365 nm UVP 3UV 8W lamp at a distance of less than 1 cm between the lamp and the sample.

[0123] Purification was performed on a Phenomenex (Torrance, California) Kinetex semi-preparative column (10 × 250 mm, 5 μm) using triethylammonium acetate (5 mM, pH 7.2) and acetonitrile as eluents on a Shimadzu (Kyoto, Japan) Prominence HPLC system. Anion exchange chromatography was performed on a Thermo Scientific (Waltham, Massachusetts) DNAPac PA-200 column (9 × 250 mm, 8 μm) using Tris (10 mM, pH 8) and NaCl (1 M) as eluents. Nucleotide analysis was performed on an Agilent 1260 HPLC system using a Kinetex Evo C18 analytical column (3.0 × 50 mm, 2.6 μm) monitored at 260 nm, using TEAA (50 mM, pH 7.2) and acetonitrile as eluents.

[0124] The DNA oligonucleotide sequence used for primer extension analysis is: ATT CAG GAC GAG CCT CAG ACC (SEQ ID NO:1)

[0125] Used in the synthesis of N3-PEG x General method of -dUTP (1 to 4)

[0126] N3-PEG x-N-hydroxysuccinimide ester (N3-PEG) x -NHS ester (4 equivalents) was dissolved in DMF (20 μL) and added to 5-propyneamino-deoxyuridine (8 μmol, 10 mM). The reaction was then stirred at room temperature in the dark for 16 hours. The reaction mixture was then purified by a semi-preparative anion exchange column (1% to 10% NaCl), evaporated under reduced pressure, and further purified on a Kinetex Evo C18 semi-preparative column (0% to 50% ACN). The resulting residue was then lyophilized to give the final product ( Figure 1A ), which is a triethylammonium salt.

[0127] N3-PEG4-dUTP(1)

[0128] Yield: 95 nmol, 44%. Electroinjection high-resolution mass spectrometry (ESI-HRMS) experimental values: m / z 793.1241; C 23 H 36 N6O 19 Calculated value of P3: (MH) - 793.1248.

[0129] N3-PEG8-dUTP(2)

[0130] Yield: 70 nmol, 35%. ESI-HRMS experimental values: m / z 969.2270; C 31 H 52 N6O 23 Calculated value of P3: (MH) - 969.2297.

[0131] N3-PEG 12 -dUTP(3)

[0132] Yield: 65 nmol, 33%. ESI-HRMS experimental values: m / z 1145.3335; C 39 H 68 N9O 27 Calculated value of P3: (MH) - 1145.3345.

[0133] N3-PEG 24 -dUTP(4)

[0134] Yield: 44 nmol, 22%. ESI-HRMS experimental values: m / z 1673.6444; C 63 H 116 N6O 39 Calculated value of P3: (MH) - 1673.6491.

[0135] N3-PEG 23 Nitrobenzyl (NB)-alcohol (5b)

[0136] In a 4mL vial containing a magnetic stir bar, add N3-PEG 23 A chilled solution of -NH2 (202 mg, 184 μmol, 1.1 equivalences) and 4-[4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy]butyric acid (50 mg, 167 μmol, 1 equivalence) in tetrahydrofuran (THF, 2 mL) was supplemented with N,N'-dicyclohexylcarbodiimide (138 mg, 669 μmol, 4 equivalences) and hydroxybenzotriazole hydrate (68 mg, 442 μmol, 2.6 equivalences). The reaction mixture was stirred at the same temperature for 5 min, and then triethylamine (116 μL, 835 μmol, 5 equivalences) was added. The reaction mixture was then heated to room temperature and allowed to stir in the dark for 16 h. The reaction mixture was then evaporated, and the residue was dissolved in acetonitrile. The resulting solid material was filtered off and purified by semi-preparative Kinetex Evo C18 column to obtain a yellow solid as the final product (170 mg, 123 μmol, 74%).

[0137] 1 H NMR(396MHz,D2O)δ7.55(s,1H),7.35(s,1H),5.44(q,J=6.3Hz,1H),4.09(t,J=6.3Hz,2H),4.00(s,3H),3.77–3.58(m ,92H),3.51(t,J=5.2Hz,2H),3.41(t,J=5.2Hz,2H),2.46(t,J=7.3Hz,2H),2.21–2.06(m,2H),1.47(d,J=6.3Hz,3H).

[0138] 13 C NMR(100MHz,D2O)δ175.28,153.61,146.31,139.07,137.49,108.87,108.67,69.74, 69.59,69.42,69.01,68.48,65.17,59.43,56.30,50.29,39.10,32.27,24.85,23.98.

[0139] ESI-HRMS experimental value: m / z 1424.7485; C 62 H 114 N5O 31 The calculated value is: (M + FA - H) - 1424.7498.

[0140] N3-PEG 23 -nitrobenzyl-dNTPs (5 to 8)

[0141] To N3-PEG 23 A solution of nitrobenzyl (NB)-ol (5b) in acetonitrile (2 mL) was added to N,N'-disuccinimidyl carbonate and 4-dimethylaminopyridine, and the mixture was stirred at 40 °C for 4 hours. The reaction was then diluted with water, and the organic material was extracted with dichloromethane. The aqueous layer was further extracted twice with dichloromethane. The combined organic layers were then washed with saturated NaHCO3, 1M HCl aqueous solution, and brine, and dried over Na2SO4. The solvent was then removed under reduced pressure, and the mixture contained N3-PEG. 23 The crude product obtained from -NB-NHS ester (compound 5c) can be used in the next step without any further purification.

[0142] The crude material containing compound 5c (2 equivalents) was dissolved in dimethylformamide (DMF, 20 μL), and 5-propynylamino-dNTP (8 μmol, 10 mM) was added to the mixture. The reaction was then stirred at room temperature in the dark for 16 hours. The reaction was then purified on a Kinetex Evo C18 semi-preparative column (0% to 50% ACN). The resulting residue was then freeze-dried to give the final product. Figure 1B ), which is a triethylammonium salt.

[0143] N3-PEG 23 -nitrobenzyl-dUTP(5)

[0144] Yield: 0.72 μmol, 7%.

[0145] 1 H NMR(400MHz,D2O)δ8.08–7.92(m,1H),7.55(s,1H),7.11(s,1H),6.28–5.96(m,2H),4.15–3.65(m,9H),3.63–3.41(m,96H) ,3.39–3.36(m,2H),3.28–3.22(m,2H),2.89(d,J=7.3Hz,3H),2.36–2.18(m,4H),2.07–1.91(m,2H),1.51(d,J=5.1Hz,3H).

[0146] 31 P NMR (162MHz, D2O) δ -10.26 (d, J = 20Hz), -11.03 (d, J = 20Hz), -22.72 (t, J = 20Hz).

[0147] ESI-HRMS experimental value: m / z 1925.7234; C 74 H 128 N8O44 Calculated value of P3: (MH) - 1925.7237.

[0148] N3-PEG 23 -nitrobenzyl-dATP(6)

[0149] Yield: 0.81 μmol, 8%.

[0150] 1 H NMR(400MHz,D2O)δ8.22(s,1H),7.75(d,J=10.5Hz,1H),7.52(d,J=33.7Hz,1H) ,7.11(d,J=14.2Hz,1H),6.58(t,J=6.7Hz,1H),6.20(s,1H),4.23(s,3H),4.14– 4.01(m,1H),3.80(s,3H),3.72–3.62(m,95H),3.59(s,4H),3.52–3.48(m,2H), 3.42–3.34(m,2H),2.67–2.43(m,2H),2.43–2.32(m,2H),1.59(t,J=6.3Hz,3H).

[0151] 31 P NMR (121MHz, D2O) δ -10.10 (d, J = 17.0Hz), -10.70 (d, J = 17.0Hz), -22.40 (t, J = 17.0Hz).

[0152] ESI-HRMS experimental value: m / z 973.3661; C 76 H 129 N 10 O 42 Calculated value of P3: (M / 2 - H) - 973.3740.

[0153] N3-PEG 23 -nitrobenzyl-dGTP(7)

[0154] Yield: 1.42 μmol, 14%.

[0155] 1H NMR(500MHz,D2O)δ7.57(s,1H),7.19(d,J=28.3Hz,2H),6.30(t,J=7.0Hz,1H),6. 19(s,1H),4.14(s,1H),4.09(d,J=5.7Hz,2H),4.00(s,3H),3.89(s,2H),3.69–3. 55(m,90H),3.53(s,4H),3.51(t,J=5.2Hz,2H),3.47–3.44(m,2H),3.32(t,J=5.4 Hz,2H),2.63–2.48(m,1H),2.37(t,J=7.1Hz,3H),2.07–2.00(m,2H),1.57(s,3H).

[0156] 31 P NMR (202MHz, D2O) δ -10.92 (d, J = 19.9Hz), -11.47 (d, J = 19.9Hz), -23.29 (t, J = 19.9Hz).

[0157] ESI-HRMS experimental value: m / z 981.3596; C 76 H 131 N 10 O 43 Calculated value of P3: (M / 2 - H) - 981.3714.

[0158] N3-PEG 23 -nitrobenzyl-dCTP(8)

[0159] Yield: 0.95 μmol, 10%.

[0160] 1 H NMR(396MHz,D2O)δ8.11(d,J=18.3Hz,1H),7.63(d,J=5.2Hz,1H),7.18(s,1H),6.25(s,1H),6.16(s,1H),4.19(d,J=11.2Hz,3H),4.06(s, 3H), 3.93 (s, 3H), 3.70–3.61 (m, 90H), 3.56 (s, 5H), 3.48 (d, J = 4.9Hz, 2H), 2.41 (t, J = 7.1Hz, 3H), 2.09–2.03 (m, 3H), 1.61 (d, J = 5.9Hz, 3H).

[0161] 31P NMR (160MHz, D2O) δ -10.34 (d, J = 20.6Hz), -11.00 (dt, J = 20.6, 19.0Hz), -22.80 (t, J = 19.0Hz).

[0162] ESI-HRMS experimental value: m / z 961.8527; C 74 H 128 N9O 43 Calculated value of P3: (M / 2 - H) - 961.8660.

[0163] Screening of TdT doping conditions

[0164] 20 μL of 6-fluorescein-labeled 21-nt oligonucleotide (200 nM), 1X TdT buffer (NEB), and TdT (2 U μL) were added to a solution. -1 CoCl2 (NEB, 0.25 mM) and nucleotides (various concentrations) were incubated at 37 °C for a specified time and quenched with an equal volume of Tris / borate / EDTA (TBE)-urea gel-loaded with dye (1X). The solution was then heated at 95 °C for 5 minutes to inactivate TdT. UV cleavage of the photoinstantaneous groups was performed using an 8W UV lamp at 365 nm for 10 minutes.

[0165] Two cycles of incorporation and deprotection

[0166] 500 μL of solution contained 6-fluorescein-labeled 21-nt oligonucleotide (200 nM), 1X TdT buffer (NEB), and TdT (2 U μL). -1 CoCl2 (NEB, 0.25 mM) and nucleotides (20 μM) were incubated at 37 °C for 7 min and quenched with EDTA (50 μL, 500 mM). The solution was concentrated to a final volume of 30 μL using a MilliporeSigma (Burlington, MA) Amicon Ultra-0.5 mL centrifuge filter (3 kDa). Sodium acetate solution (10 μL, 3 M, pH 5.0) was added to the concentrated solution, and further purification was performed using a Zymo (Irvine, California) DNA Cleaning and Concentration Kit. UV cleavage of the photostable groups was performed using an 8W UV lamp at 365 nm for 10 min. This step was then repeated to obtain the second incorporation.

[0167] denaturing PAGE gel

[0168] Prewash Initrogen (Carlsbad, California) Novex with 1X TBE run buffer.TM TBE-urea gel (15%) was used to remove excess urea. An oligonucleotide sample (5 μL) was mixed with Novex. TM The TBE-urea sample buffer (2X, 5 μL) was mixed. The mixture was then heated at 95°C for 5 minutes and loaded onto a gel. The gel was run in 1X TBE run buffer at up to 240V until bromophenol blue reached the bottom of the gel. The gel was then visualized using Gel Doc XR+ and analyzed using the gel analysis tool in Bio-Rad (Hercules, California) Image Lab software.

[0169] Results and Discussion

[0170] To investigate the effect of PEG chain length on TdT incorporation, PEGs of various lengths derived from repeating units of ethylene glycol, ranging from four to twenty-four, were conjugated to propargylamino-dUTP. This resulted in azide-PEG. x -NHS esters (x = 4, 8, 12, 24) are reacted with propargylamino-dUTP to give azide-PEG. x -dUTP (where x = 4, 8, 12, 24) (Figure 1). The reaction yield changed from 22% to 48%.

[0171] Then compounds 1 to 4 were used for incorporation assays using TdT enzymes. Figures 2A to 2B Using 40 μM nucleotides, 200 nM 21-nt oligonucleotides for extension, and 1 U μL -1 At TdT, we observed various yields of 22-nt oligonucleotides (+1 product). When using the shorter compound 1 (PEG4), 22-nt (+1 product) was formed in 55% yield after 15 minutes, along with a significant amount of 23-nt (+2 product) formed in 41% yield and some 24-nt oligonucleotides (+3 product) formed in 4% yield (lane 1). Compared with the shorter PEG4 modification, incorporation with compound 2, which has a PEG8-terminated group, gave a much higher yield of the +1 product (88% yield) (lane 2). Furthermore, the yield of the +2 product was significantly reduced (12% yield), and no +3 product was observed. Further increasing the PEG length to 12 repeating units (compound 3) showed better selectivity with a +1 product yield of 94% (lane 3), and doubling the PEG length to 24 repeating units (compound 4) gave the desired 22-nt oligonucleotides (+1 product) in quantitative yield (lane 4). Furthermore, the incorporation of propargylamino-dUTP is significantly slower compared to the incorporation of dTTP. (Lane 6 to 7) Figure 2AUnbound by any particular theory, the presence of hydrophobic molecular scars may slow down TdT activity. After incubating the mixture for 30 minutes, the increase in the yield of the undesired 23-nt oligonucleotide increased (+2 product). Compound 1 gave a lower yield of 22-nt oligonucleotide (+1 product, 35%), while increasing the yields of the undesired 23-nt (+2 product, 56%) and 24-nt (+3 product, 9%) oligonucleotides. For compounds 2, 3, and 4, no 24-nt oligonucleotide (+3 product) was observed. However, an increase in the yield of 23-nt oligonucleotide was observed in compounds 2 (+2 product, 22%) and 3 (+2 product, 13%) compared to the yield obtained with a 15-minute incubation. In compound 4, only a small amount of 23-nt oligonucleotide (+2 product) (2%) was observed.

[0172] Longer PEG chains, with base-capped ends, yielded higher yields of 22-nt oligonucleotides (+1 product), or single incorporation events. While PEG4 yielded 55% after 15 minutes of incubation, PEG... 24 A quantitative incorporation was observed. A similar trend was observed after 30 minutes of incubation, which was consistent with when PEG was used. 24 Compared to the 98% yield of the previous method, PEG4 yielded 35%. These results show that the length of the PEG can be adjusted to achieve optimal incorporation efficiency.

[0173] Figure 2A Lanes 1-4: Denatured TBE-urea gels incorporating compounds 1-4. Lanes 1-4: Incorporation of compounds 1-4 (40 μM) for 15 minutes each. Lanes 5 and 12: 21-nt oligonucleotides. Lane 6: Incorporation of propargylamino-dUTP (20 μM). Lane 7: Incorporation of dTTP (20 μM). Lanes 8-11: Incorporation of compounds 1-4 (40 μM) for 30 minutes each. Figure 2B : A bar graph showing the effect of PEG length on the yield of 22-nt oligonucleotides.

[0174] Based on these results, PEG can be photolyzed using a linker. 23 Partially linked to dNTPs (Scheme 1). First, in amino-functionalized PEG 23 An amide bond was formed between the compound and 4-[4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy]butyric acid to give compound 5b in 77% yield. To further conjugate photolyzable PEG with propargylamino-dUTP, 5b was treated with N,N'-disuccinimidyl carbonate, and the resulting carbonate 5c was further coupled with propargylamino-dUTP to give the final product 5 in 7.2% yield.

[0175] Then the purified photolyzable PEG 23 -dUTP 5 is used for incorporation via TdT. Optimization of reaction conditions showed that quantitative incorporation was achieved with compound 5 within a reaction time of 7 minutes. Figure 3 ). Figure 3 Single selective incorporation of compound 5 was observed at 7 minutes of incubation, with incorporation time increasing from 5 minutes to 9 minutes in lanes 1 to 5. Lanes 10 to 14 show the incorporation in PEG. 23 +1 incorporation following photolysis of nucleotide end caps.

[0176]

[0177] Scheme 1. Synthesis of compound 5.

[0178] To compare the incorporation of base-modified nucleotides and 3'-azidomethyl-terminated dTTP, PEGylation via TdT was performed. 23 The incorporation of -PC-dUTP and 3'-O-azidomethyl-dTTP was compared. By changing the concentration of 3'-O-azidomethyl-dTTP from 1 μM to 32 μM, TdT could not be incorporated into the 3'-terminated nucleotide after 10 minutes of incubation. Figure 4 On the other hand, base-modified nucleotides can be incorporated at a concentration of 20 μM (lane 10, Figure 4 This result shows that TdT can be advantageously incorporated into base-modified nucleotides compared to 3'OH-modified nucleotides. Figure 4 After incubation at 37°C for 10 minutes, no incorporation of 3'-O-azidomethyl-dTTP nucleotides was observed. Lanes 1 and 8: 21-nt primers. Lanes 2 to 7: Increased nucleotide concentrations (1 to 32 μM). Lane 9: Incorporation of 1 μM dTTP as a positive control. Lane 10: PEG. 23 Incorporation of -PC-dUTP.

[0179] With optimized conditions, the suitability of using PEG as a capping group for DNA synthesis was tested using two cycles of incorporation and deprotection. Using compound 5, we incorporated the first nucleotide (lane 2), followed by TdT inactivation. Photoinduced cleavage of the PEG moiety yielded a 22-nt product (+1 product, lane 3). The oligonucleotide was then purified to remove nucleotides, salts, and TdT. TdT incorporation of the second nucleotide (compound 5) yielded the desired product (lane 4), followed by PEG moiety removal to obtain the desired 23-nt oligonucleotide (+2 product, lane 5). The resulting 22-nt (+1 product) and 23-nt (+2 product) oligonucleotides are placed side-by-side to show the final product after each incorporation cycle (lanes 6 to 8). Figure 5 ) Figure 5 Two cycles of incorporation and deprotection were performed to demonstrate the feasibility of using compound 5 for the synthesis of enzymatic oligonucleotides. Lane 1: 21-nt oligonucleotide. Lane 2: Incorporation of the first nucleotide. Lane 3: Photolysis of the PEG moiety. Lane 4: Incorporation of the second nucleotide. Lane 5: Photolysis of the PEG moiety. Lanes 6 to 8: Lanes 1, 3, and 5, respectively.

[0180] Since the oligonucleotide cleaning kit is only about 80% efficient, the reduction in oligonucleotide concentration after cleaning will affect the yield of the second incorporation. This loss explains the strength difference observed in lanes 5 and 8. This loss can be reduced by using a solid support (such as glass or magnetic beads), resulting in a cleaner product at each cycle.

[0181] The total time for each cycle, including the 7 minutes required for incorporation and the 10 minutes for deprotection, plus the time needed to purify the oligonucleotide, is approximately 30 minutes. Using a higher-powered UV lamp or other cleavable chemicals (such as disulfide bonds with faster kinetic rates) can allow for faster cleavage of the PEG nucleobase capping. The use of a solid support also reduces the need for purification, further reducing the time required per cycle.

[0182] Following the successful single incorporation of nucleobase-modified dUTP via TdT, the synthesis of modified dATP, dGTP, and dCTP (compounds 6-8) was carried out, providing yields in the range of 7% to 14% (Scheme 2). The nucleotides were then purified by high-performance liquid chromatography (HPLC) using a C18 column to obtain products of high purity. Because these compounds are highly unstable upon exposure to light, they were immediately transferred to LightSafe microcentrifuge tubes after purification for lyophilization and storage.

[0183]

[0184] Scheme 2: Synthesis of compounds 5 to 8.

[0185] Compounds 5 through 8 were then used for TdT incorporation, using 80 μM compound 5 and 40 μM compounds 6 through 8. For all nucleotides, incorporation yields exceeding 88% were obtained. Figure 6 The yield can be further improved by optimizing the nucleotide concentration to achieve yields comparable to the phosphoramidite method. Figure 1 shows the incorporation of nucleotides 5 to 8 via TdT. Lanes 1, 6, and 11: 21-nt oligonucleotides. Lanes 2 to 5: Incorporation of compounds 7, 6, 8, and 5, respectively. Lanes 7 to 10: Photolysis of the products in lanes 2 to 5, respectively.

[0186] To reduce the time required for photolysis of PEG chains, the cleavage efficiency using various UV lamps was compared. A 40W UV lamp was used in comparison to an 8W UV lamp. Using 200 μM of protected nucleotides, the nucleotides were exposed to 370 nm UV light at the 40W lamp for 1 to 3 minutes, and then exposed to 365 nm UV light at the 8W lamp for 5 minutes. The nucleotides were then protected from light and analyzed. Using the 40W lamp, over 99% cleavage was observed after 1 minute, and no further cleavage of the protected nucleotides was observed after 3 minutes of exposure. Using the 8W lamp, only 56% cleavage of the photoinstantaneous nucleotides was achieved. Therefore, oligonucleotides with specific sequences can be synthesized with each cycle taking less than 10 minutes, including 7 minutes of incorporation and 1 minute of photolysis.

[0187] A systematic study of the base capping size requirements for nucleotides used in single incorporation events revealed that discrete polyethylene glycol chains of 23 monomeric ethylene glycol units can act as capping groups on the nucleobases of nucleotide triphosphates, enabling single nucleotide incorporation using commercially available TdT. Subsequent incorporation is achieved during the cleavage of the capping groups.

[0188] Other considerations include the presence of the propargyl amino moiety (also known as a molecular scar), which may slow down the incorporation efficiency. The incorporation of propargyl amino-dUTP is slower than that of dTTP (the natural substrate of TdT) (lanes 6 and 7). Figure 2A This can reduce the time required for incorporation and removal of the PEG moiety, thereby lowering the total time per cycle. Finally, it can improve the incorporation yield to match or even exceed the yields achieved by the phosphoramidite method for longer oligonucleotide synthesis. Other nucleobase chemistry can enable faster removal of nucleobase modifications without leaving molecular scars.

[0189] the term

[0190] In at least some of the foregoing embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, unless such substitution is technically not feasible. Those skilled in the art will understand that various other omissions, additions, and modifications may be made to the above methods and structures without departing from the scope of the claimed subject matter. All such modifications and alterations are intended to fall within the scope of the subject matter defined by the appended claims.

[0191] Regarding the use of substantially any plural and / or singular terms herein, those skilled in the art can appropriately convert them from plural to singular and / or from singular to plural depending on the context and / or application. For clarity, various singular / plural arrangements may be explicitly shown herein. As used in this specification and the appended claims, the singular forms “a,” “an,” and “described” include plural referents unless the context clearly specifies otherwise. Unless otherwise indicated, any reference to “or” herein is intended to include “and / or.”

[0192] Those skilled in the art will understand that, in general, the terminology used herein, particularly in the appended claims (e.g., the body of the appended claims), is intended to be “open-ended” (e.g., the term “comprising” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “at least having,” the term “including” should be interpreted as “comprising but not limited to,” etc.). Those skilled in the art will also understand that if a specific number of claims is intentional, this intention will be explicitly stated in the claims, and if such a number is not present, this intention is not present. For example, to aid understanding, the appended claims may include the use of the introductory phrases “at least one” and “one or more” to introduce the claims. However, even when the same claim includes the introductory phrase "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"), the use of such phrases should not be construed as implying that introducing a claim statement with the indefinite article "a" or "an" limits any particular claim containing such an introduced claim statement to an embodiment containing only one such statement; the same applies to the use of definite articles to introduce claim statements. Furthermore, even when a specific number of introduced claim statements is explicitly stated, those skilled in the art will recognize that such a statement should be interpreted as meaning at least the number stated (e.g., in the absence of other modifiers, a direct statement of "two statements" means at least two statements, or two or more statements). Furthermore, in cases where conventions such as "at least one of A, B, and C" are used, generally speaking, this convention is intended to be understood by those skilled in the art to be used in the sense of the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having a single A, a single B, a single C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In cases where conventions such as "at least one of A, B, or C" are used, generally speaking, this convention is intended to be understood by those skilled in the art to be used in the sense of the convention (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, systems having a single A, a single B, a single C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art should also understand that, in fact, any transitional words and / or phrases presenting two or more alternative terms in the specification, claims, or drawings should be understood to contemplate the possibility of including one of the terms, any one of the terms, or both of the terms. For example, the phrase “A or B” would be understood to include the possibility of “A” or “B” or “A and B”.

[0193] Furthermore, when features or aspects of this disclosure are described in terms of the Markush group, those skilled in the art will recognize that this disclosure is also described in terms of any single member or subgroup of the Markush group.

[0194] As those skilled in the art will understand, for any and all purposes, such as for the purpose of providing a written description, all scopes disclosed herein also include any and all possible subscopes and combinations thereof. Any listed scope can be readily identified as sufficiently descriptive and such that the same scope can be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein can be readily decomposed into a lower third, middle third, and upper third, etc. As those skilled in the art will also understand, all language such as “at most,” “at least,” “greater than,” “less than,” etc., includes the referenced numbers and refers to a scope that can subsequently be decomposed into subscopes as described above. Finally, as those skilled in the art will understand, a scope includes each individual member. Thus, for example, a group with 1-3 clauses means a group with 1, 2, or 3 clauses. Similarly, a group with 1-5 clauses means a group with 1, 2, 3, 4, or 5 clauses, etc.

[0195] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The aspects and embodiments disclosed herein are for illustrative purposes and are not intended to be limiting, wherein the true scope and spirit are indicated by the following claims.

Claims

1. A method for nucleic acid synthesis, the method comprising: (a1) Provides a nucleic acid, a first nucleoside triphosphate and a first terminal deoxynucleotidyl transferase (TdT), wherein the first nucleoside triphosphate comprises a modified base, the modified base comprising a photolyzable carbon chain moiety having a length of at least 60 Å; (b1) Contact (i) the nucleic acid and (ii) the first nucleoside triphosphate with the first TdT to generate a first modified nucleic acid, the first modified nucleic acid comprising the nucleic acid incorporated with a first nucleotide, the first nucleotide comprising the modified base from the first nucleoside triphosphate; as well as (c1) Photolyzing the modified base of the first nucleotide in the photolyzable carbon chain portion of the first modified nucleic acid to remove the photolyzable carbon chain portion from the first modified nucleic acid.

2. The method according to claim 1, further comprising: (a2) Provide a second nucleoside triphosphate and a second TdT, wherein the second nucleoside triphosphate comprises a modified base, the modified base comprising a photolyzable carbon chain moiety having a length of at least 60 Å; (b2) Contacting (i) the first modified nucleic acid in which the photolytic carbon chain portion of the modified base of the first nucleotide is removed and (ii) the second nucleoside triphosphate with the second TdT to generate a second modified nucleic acid, the second modified nucleic acid comprising the first modified nucleic acid incorporated with a second nucleotide, the second nucleotide comprising the modified base from the second nucleoside triphosphate; as well as (c2) Photolyzing the modified base of the second nucleotide in the photolyzable carbon chain portion of the second modified nucleic acid to remove the photolyzable carbon chain portion from the second modified nucleic acid.

3. A method for nucleic acid synthesis, the method comprising: (a) Provides a nucleic acid and a plurality of nucleoside triphosphates, wherein each of the plurality of nucleoside triphosphates comprises a modified base, the modified base comprising a photolyzable carbon chain moiety having a length of at least 60 Å; (b1) Contact the nucleic acid of (i) and the first nucleoside triphosphate of the plurality of nucleoside triphosphates of (ii) with a first terminal deoxynucleotidyl transferase (TdT) to generate a first modified nucleic acid, the first modified nucleic acid comprising the nucleic acid incorporated with a first nucleotide, the first nucleotide comprising the modified base from the first nucleoside triphosphate; (c1) Photolyzing the modified base of the first nucleotide in the photolyzable carbon chain portion of the first modified nucleic acid to remove the photolyzable carbon chain portion from the first modified nucleic acid; (b2) Contacting (i) the first modified nucleic acid in which the photolytic carbon chain portion of the modified base of the first nucleotide is removed and (ii) the second nucleoside triphosphate of the plurality of nucleoside triphosphates with a second TdT to generate a second modified nucleic acid, the second modified nucleic acid comprising the first modified nucleic acid incorporated with a second nucleotide, the second nucleotide comprising the modified base from the second nucleoside triphosphate; as well as (c2) Photolyzing the modified base of the second nucleotide in the photolyzable carbon chain portion of the second modified nucleic acid to remove the photolyzable carbon chain portion from the second modified nucleic acid.

4. The method according to any one of claims 1 to 3, wherein the concentration of the nucleic acid is at least 10 nM.

5. The method according to any one of claims 1 to 3, wherein the nucleic acid comprises a single-stranded (ss) nucleic acid.

6. The method according to any one of claims 1 to 3, wherein the nucleic acid comprises deoxyribonucleic acid (DNA).

7. The method according to any one of claims 1 to 3, wherein the nucleic acid comprises at least one ribonucleotide.

8. The method according to any one of claims 1 to 3, wherein the nucleic acid is ligated to a solid carrier.

9. The method according to claim 8, wherein the solid carrier comprises a flow cell.

10. The method of claim 8, further comprising separating the modified nucleic acid from the solid carrier.

11. The method according to any one of claims 1 to 3, wherein the concentration of the first nucleoside triphosphate and / or the second nucleoside triphosphate is at least 0.1 µM.

12. The method according to any one of claims 1 to 3, wherein the modified base comprises modified cytosine, modified uracil, modified thymine, modified adenine, or modified guanine.

13. The method according to any one of claims 1 to 3, wherein the modified base comprises propargylamino group, aminoallyl group, propargylhydroxy group or a combination thereof.

14. The method according to any one of claims 1 to 3, wherein the photolyzable carbon chain portion comprises saturated or unsaturated, substituted or unsubstituted, straight or branched carbon chains.

15. The method of claim 14, wherein the carbon chain has a length of at least 60 Å.

16. The method of claim 14, wherein the photodegradable carbon chain portion comprises at least 54 carbon atoms, oxygen atoms, nitrogen atoms and / or sulfur atoms in the main chain of the carbon chain.

17. The method according to any one of claims 1 to 3, wherein the photolytic carbon chain portion comprises a plurality of repeating units.

18. The method of claim 17, wherein the plurality of repeating units comprises the same repeating unit.

19. The method of claim 17, wherein one of the plurality of repeating units comprises at least three carbon atoms, oxygen atoms, nitrogen atoms and / or sulfur atoms in the main chain of the repeating unit.

20. The method of claim 17, wherein the plurality of repeating units comprise polyethylene glycol (PEG).

21. The method of claim 17, wherein the repeating units in the plurality of repeating units do not include aromatic groups.

22. The method of claim 17, wherein the repeating unit in the plurality of repeating units comprises an aromatic group.

23. The method of claim 17, wherein the number of the plurality of repeating units is at least 18.

24. The method according to any one of claims 1 to 3, wherein the photolyzable carbon chain portion comprises a photolyzable portion selected from the group consisting of: carbonyl group, arylcarbonylmethyl group, benzoylmethyl group, o-alkylbenzoylmethyl group, p-hydroxybenzoylmethyl group, diphenylethanol ketone group, benzyl group, nitroaryl group, nitrobenzyl group, o-nitrobenzyl group, o-nitro-2-phenylethyloxycarbonyl group, o-nitroaniline, coumarin-4-ylmethyl group, arylmethyl group, coumarin group, o-hydroxyarylmethyl group, metal-containing group, neopentylyl group, ester of carboxylic acid, arylsulfonyl group, Ketone groups, carbon-negative mediated groups, silyl groups, silyl groups, 2-hydroxycinnamyl groups, α-ketoamide groups, α,β-unsaturated aniline, methyl (phenyl)thiocarbamate groups, S,S-sulfur dioxide chromone groups, 2-pyrrolidine-1,4-benzoquinone groups, triazine groups, arylmethylene imino groups, xanthracene groups, pyronin groups, 7-hydroxy-1,1-dimethylnaphthone groups, carboxylic acid groups, phosphate groups, phosphite groups, sulfate groups, acid groups, alcohol groups, thiol groups, N-oxide groups, phenolic groups, amine groups, derivatives of any of the above substances, or combinations thereof.

25. The method according to any one of claims 1 to 3, wherein the concentration of the first TdT and / or the second TdT is at least 10 nM.

26. The method according to any one of claims 1 to 3, wherein the first TdT and / or the second TdT comprises recombinant TdT.

27. The method according to any one of claims 1 to 3, wherein the first TdT and the second TdT are the same.

28. The method according to any one of claims 1 to 3, wherein the first TdT and the second Tdt comprise the same molecule of TdT.

29. The method according to any one of claims 1 to 3, wherein the first TdT and the second Tdt comprise different molecules of TdT.

30. The method according to any one of claims 1 to 3, wherein the first TdT and the second TdT are different TdTs.

31. The method according to any one of claims 1 to 3, further comprising: Remove the first TdT after step (b1) and before step (c1); And the second TdT is removed after step (b2) and before step (c2).

32. The method according to claim 31, Wherein the first TdT is connected to the first magnetic bead, and the removal of the first TdT includes magnetic removal of the first TdT after step (b1) and before step (c1), and / or The second TdT is connected to the second magnetic bead, and the removal of the second TdT includes magnetic removal of the second TdT after step (b2) and before step (c2).

33. The method of claim 32, wherein the first magnetic bead and the second magnetic bead are identical.

34. The method according to any one of claims 1 to 3, further comprising: The first TdT is deactivated after step (b1) and before step (c1); And the second TdT is deactivated after step (b2) and before step (c2).

35. The method of claim 34, wherein deactivating the first TdT comprises thermally deactivating the first TdT, and wherein deactivating the second TdT comprises thermally deactivating the second TdT.

36. The method according to any one of claims 1 to 3, The contact time in step (b1) is 5 to 20 minutes, and The contact time in step (b2) is 5 to 20 minutes.

37. The method according to any one of claims 1 to 3, The contact described in step (b1) is performed at temperatures ranging from 16°C to 58°C, and The contact step (b2) is performed at a temperature between 16°C and 58°C.

38. The method according to any one of claims 1 to 3, The first modified nucleic acid in step (b1) comprises at least 95% of the nucleic acid, and The second modified nucleic acid in step (b2) comprises at least 95% of the first modified nucleic acid.

39. The method according to any one of claims 1 to 3, In step (b1), at least 95% of the first modified nucleic acid comprises the first modified nucleic acid, which includes the nucleic acid incorporating a single first nucleotide from the first nucleoside triphosphate, and In step (b2), at least 95% of the second modified nucleic acid comprises the second modified nucleic acid, which comprises the first modified nucleic acid incorporating a single second nucleotide from the second nucleoside triphosphate.

40. The method according to any one of claims 1 to 3, The photolysis described in step (c1) is performed using the first radiation, and The photolysis in step (c2) is performed using a second radiation.

41. The method according to any one of claims 1 to 3, wherein the first radiation and / or the second radiation has a power of 5 watts to 20 watts.

42. The method of claim 40, wherein the first radiation and / or the second radiation comprises radiation having ultraviolet radiation.

43. The method of claim 40, wherein the first radiation and / or the second radiation comprises radiation having a wavelength of 300 nm to 400 nm.

44. The method of claim 40, wherein an ultraviolet (UV) lamp with a power of 10 watts to 60 watts is used to generate the first radiation and / or the second radiation.

45. The method according to any one of claims 1 to 3, wherein the photolysis in step (c1) and / or the photolysis in step (c2) is performed for 1 to 20 minutes.

46. ​​The method according to any one of claims 1 to 3, wherein the photolysis in step (c1) and / or step (c2) has an efficiency of at least 90%.

47. The method according to any one of claims 1 to 3, wherein the contact in step (b1) and the contact in step (b2) are each completed within 7 minutes.

48. The method according to any one of claims 1 to 3, wherein the photolysis in step (c1) and the photolysis in step (c2) are each completed within 1 minute.

49. The method according to any one of claims 1 to 3, wherein the contact in step (b1) and the photolysis in step (c1) are completed within 10 minutes, and wherein the contact in step (b2) and the photolysis in step (c2) are completed within 10 minutes.

50. The method according to any one of claims 1 to 3, further comprising: Polymerase is used to generate the reverse complementary sequence of the modified nucleic acid.

51. A method for synthesizing nucleic acids, the method comprising: (a1) Provide nucleic acid, Iteratively, (a2) Provides a plurality of nucleoside triphosphates and terminal deoxynucleotidyl transferase (TdT), wherein the nucleoside triphosphate comprises a modified base, the modified base comprising a photolyzable carbon chain moiety having a length of at least 60 Å; (b) Contacting (i) the nucleic acid in (a1) of the first iteration or the modified nucleic acid in (c) of any iteration other than the first iteration, and (ii) the nucleoside triphosphate, with the TdT to generate a modified nucleic acid comprising a nucleotide incorporated in the nucleic acid in (a1) of the first iteration or the modified nucleic acid in (c) of any iteration other than the first iteration, the nucleotide comprising the modified base from the nucleoside triphosphate; as well as (c) Photolyzing the modified bases of the nucleotide in the photolyzable carbon chain portion of the modified nucleic acid to remove the photolyzable carbon chain portion from the modified nucleic acid. This generates the modified nucleic acid, which includes a predetermined sequence.

52. The method of claim 51, wherein at least 95% of the modified nucleic acids generated after multiple iterations comprise the predetermined sequence.

53. The method of claim 52, wherein the multiple iterations comprise at least 200 iterations.

54. The method according to any one of claims 51 to 53, the method comprising: Receive the predetermined sequence.

55. A plurality of nucleoside triphosphates for use in nucleotide synthesis of terminal deoxynucleotidyl transferase (TdT), each nucleoside triphosphate comprising a modified base, wherein the modified base comprises a photolyzable carbon chain moiety having a length of at least 60 Å.

56. The plurality of nucleoside triphosphates according to claim 55, wherein the modified bases include modified cytosine, modified uracil, modified thymine, modified adenine, or modified guanine.

57. A plurality of nucleoside triphosphates according to any one of claims 55 to 56, wherein the modified base comprises an acetylacetylamino group, an aminoallyl group, or a combination thereof.

58. A plurality of nucleoside triphosphates according to any one of claims 55 to 56, wherein the photolyzable carbon chain portion comprises saturated or unsaturated, substituted or unsubstituted, straight or branched carbon chains.

59. The plurality of nucleoside triphosphates according to claim 58, wherein the carbon chain has a length of at least 60 Å.

60. The plurality of nucleoside triphosphates according to claim 58, wherein the photolyzable carbon chain portion comprises at least 54 carbon atoms, oxygen atoms, nitrogen atoms and / or sulfur atoms in the main chain of the carbon chain.

61. A plurality of nucleoside triphosphates according to any one of claims 55 to 56, wherein the photolyzable carbon chain portion comprises a plurality of repeating units.

62. The plurality of nucleoside triphosphates according to claim 61, wherein the plurality of repeating units comprise the same repeating unit.

63. The plurality of nucleoside triphosphates according to claim 61, wherein one of the plurality of repeating units comprises at least three carbon atoms, oxygen atoms, nitrogen atoms and / or sulfur atoms in the main chain of the repeating unit.

64. The plurality of nucleoside triphosphates according to claim 61, wherein the plurality of repeating units comprises polyethylene glycol (PEG).

65. The plurality of nucleoside triphosphates according to claim 61, wherein the repeating unit in the plurality of repeating units does not include an aromatic group.

66. The plurality of nucleoside triphosphates according to claim 61, wherein the repeating unit in the plurality of repeating units comprises an aromatic group.

67. The plurality of nucleoside triphosphates according to claim 61, wherein the number of the plurality of repeating units is at least 18.

68. The plurality of nucleoside triphosphates according to claim 61, wherein the photolyzable carbon chain portion comprises a photolyzable portion selected from the group consisting of: carbonyl group, arylcarbonylmethyl group, benzoylmethyl group, o-alkylbenzoylmethyl group, p-hydroxybenzoylmethyl group, diphenylethanol ketone group, benzyl group, nitroaryl group, nitrobenzyl group, o-nitrobenzyl group, o-nitro-2-phenylethyloxycarbonyl group, o-nitroaniline, coumarin-4-ylmethyl group, arylmethyl group, coumarin group, o-hydroxyarylmethyl group, metal-containing group, neopentylyl group Groups, esters of carboxylic acids, aryl sulfonyl groups, ketones, carbon-negative mediated groups, silyl groups, silyl groups, 2-hydroxycinnamyl groups, α-ketoamides, α,β-unsaturated aniline, methyl (phenyl)thiocarbamate, S,S-sulfur chromone, 2-pyrrolidine-1,4-benzoquinone groups, triazine groups, aryl methylene imino groups, xanthracene groups, pyronin groups, 7-hydroxy-1,1-dimethylnaphthone, carboxylic acids, phosphates, phosphites, sulfates, acids, alcohols, thiols, N-oxides, phenols, amines, derivatives of any of the above substances, or combinations thereof.

69. The plurality of nucleoside triphosphates according to claim 68, wherein the photolytic portion is photolytically decomposed by radiation of power of 5 watts to 20 watts within 1 to 20 minutes with an efficiency of at least 90%.

70. The plurality of nucleoside triphosphates according to claim 69, wherein the radiation includes ultraviolet (UV) radiation.

71. The plurality of nucleoside triphosphates according to claim 68, wherein the radiation has a wavelength of 300 nm to 400 nm.

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