Use of 3'-oxymethylenealkyldisulfide-protected nucleotides for enzymatic DNA and RNA synthesis.

Abstract

The present invention relates to the enzymatic synthesis of nucleic acids using 3'-O-(CH2SSR) protected reversible nucleotide terminators. The nucleotide bases of the protected nucleotides can be natural or non-natural (e.g., 7-deazaG and 7-deazaA), or mixtures thereof. The bases of the nucleotides can be modified with linkers bearing carboxylic acid (-CO2H), amine (-NH2), thiol (-SH), hydroxymethyl (-CH2OH), propargylamine, alkyne groups, etc., for subsequent modification and labeling. Such nucleotide substrates can be incorporated into single-stranded, template-free nucleic acids, or template DNA hybrids. Then, in a later step, the 3'-O-CH2SSR protecting group can be cleaved by a chemical treatment, which is a prerequisite for enzymatic nucleic acid synthesis. Starting from short seeded DNA strands, longer DNA strands can be synthesized in solution or on a solid surface by adding nucleotides in a defined manner and carrying out a cleavage reaction after each step.

Description

[Technical field]

[0001] Background technology The technologies of DNA sequencing and DNA synthesis are fundamental to modern biology: DNA sequencing allows us to read the genetic code, while DNA synthesis allows us to manufacture genes and produce their products in the laboratory.

[0002] Of these, the latter is lagging far behind in many aspects. Massively parallel DNA sequencing methods have more significantly surpassed DNA synthesis methods since the past two decades with the introduction of next-generation DNA sequencing (NGS) technologies.

[0003] Meanwhile, DNA synthesis methods have remained essentially unchanged for the past few decades. DNA synthesis is required in many areas of biology, from developing diagnostic kits, to manufacturing active pharmaceutical ingredients (APIs) for nucleic acid therapeutics, to building DNA-based data storage systems. Currently, nucleic acid synthesis is primarily performed on solid supports using phosphoramidite chemistry, which was developed in 1981 by Marvin Caruthers and his colleagues at the University of Colorado.

[0004] The phosphoramidite method is not environmentally friendly because the reaction occurs in an organic phase, and a significant amount of organic waste is generated. Furthermore, this method can only produce short nucleic acids, with a limit of about 200 nucleotides. On the other hand, most gene constructs require lengths in the range of 2,000 to 3,000 nucleotides. To obtain oligonucleotides longer than this, the only viable method so far is a PCR reaction using short synthetic nucleic acids with overlapping segments to be spliced ​​together and amplified in a stepwise manner to generate long DNA molecules. In many cases, such methods are time-consuming and expensive.

[0005] To overcome this limitation, considerable efforts have been made to develop alternative methods for DNA synthesis, primarily due to the need to achieve rapid, accurate and efficient synthesis of longer DNA molecules, the ability to generate large numbers of DNA sequences simultaneously, environmental friendliness, and overall cost reduction.

[0006] One method that has attracted great interest in this regard is template-free DNA synthesis using reversibly terminating nucleotides and the TdT enzyme. In this method, nucleotides are added one at a time and terminated by cleavage after one extension. This requires nucleotides with a capping group on the 3'-OH (WO 2016 / 128731, US 10,774,316). Another method that works in a similar way involves the use of nucleotides directly linked to TdT via a cleavable linker (Nature Biotechnology, vol 36, pages 645-650, 2018). In both cases, the first step involves the addition of nucleotides in a defined manner, terminating DNA synthesis by incorporation of a single base. Then, to make the growing chain available for incorporation of a subsequent base, a chemical treatment regenerates the n+1 strand.

[0007] This is very similar to how the most powerful DNA sequencing methods work, often known as sequencing by synthesis (SBS). In both cases, the 3'-OH group of the nucleotide needs to be capped with a smaller functional group in order to be enzymatically incorporated. More importantly, the capping group must be removed by chemical treatment under conditions suitable for fragile DNA molecules. Thus, reversible nucleotide terminators that have been demonstrated to be useful for sequencing by synthesis are likely to also fulfill two fundamental requirements for enzymatic DNA synthesis: termination by a single base and cleavability under DNA-compatible conditions.

[0008] It is known that 3'-OH protected nucleotides, especially 3'-O-CH2SSMe, are accepted by polymerases due to their small size and terminate DNA synthesis by incorporation of a single base. In a subsequent step, they can be removed using mild chemicals such as thiols or phosphines. Such techniques are disclosed in US Patents 10,301,346, 10,273,539 and 10,336,785 and are used with template oligonucleotides in the sequencing process.

[0009] Many nucleotide classes are good substrates for enzymatic incorporation into growing DNA chains, but are not cleavable under mild conditions compatible with DNA, limiting their use in DNA sequencing. Such nucleotides are not an option for direct, i.e. template-free, synthesis of DNA or RNA, because cleavability under conditions compatible with DNA or RNA is an essential aspect of both DNA sequencing and synthetic methods.

[0010] It was therefore an object of the present invention to provide blocked nucleotides for the direct synthesis of DNA and RNA by enzymatic reactions.

[0011] overview We found that 3'-OH protected nucleotides, especially 3'-O-CH2SSMe, known from the sequencing-by-synthesis process, can be used for direct template-free enzymatic synthesis of DNA and RNA.

[0012] A subject of the present invention is therefore a method for the synthesis of a DNA or RNA strand, comprising: a) providing a primer capable of binding to a nucleotide; b) General formula (I) [ka] [In the formula, B=natural or unnatural nucleobase; R=-Me, -Et, -Pr, -iPr, -iBu, CH2CH2NH2, CH2CH2CN, providing a first nucleotide with R1, R2=H, D, -Me, -Et, -Pr, -iPr, -iBu, CHF2CH2CH2F; c) providing a terminal transferase to ligate the first nucleotide to the primer; d) cleaving and removing the protecting group SS-R from the first nucleotide; e) repeating steps b) to d) by providing further nucleotides according to general formula (I) to obtain a DNA or RNA strand. The method includes:

[0013] The method of the present invention is not directed to obtaining the sequencing information of DNA or RNA strand, but to the method of synthesizing DNA or RNA strand.Therefore, in a preferred embodiment, the first and further nucleotide according to general formula (I) provided in step b) and e) does not contain detectable label.The term "detectable label" includes all the labels used in the technology of sequencing by synthesis, such as dyes, especially fluorescent dyes, mass tags and radioactive tags.

[0014] Theoretically, steps b)-d) (i.e., step e)) can be repeated as many times as necessary to obtain a DNA or RNA strand having the desired length, but in practice, steps b)-d) (i.e., step e)) are usually repeated 10 to 1000 times.

[0015] Nucleotides bearing a 3'-OCH2SSMe group can terminate DNA synthesis by a single base extension and are free to be further extended when treated with TCEP or thiol compounds. TCEP and thiol compounds are mild and work well under DNA-compatible conditions, making them good candidates for DNA and RNA synthesis.

[0016] The method of the present invention allows the enzymatic synthesis of nucleic acids using 3'-O-(CH2SSR) protected reversible nucleotide terminators. The nucleotide bases of the protected nucleotides can be natural or unnatural (e.g., 7-deazaG and 7-deazaA), or mixtures thereof. The nucleotide bases can be modified with linkers bearing carboxylic acid (-CO2H), amine (-NH2), thiol (-SH), hydroxymethyl (-CH2OH), propargylamine, alkyne groups, etc., for subsequent modification and labeling. Such nucleotide substrates can be incorporated into single-stranded, template-free nucleic acids, or template DNA hybrids. Then, in a later step, the 3'-O-CH2SSR protecting group can be cleaved by a chemical treatment, which is a prerequisite for enzymatic nucleic acid synthesis. Starting from short seeded DNA strands, longer DNA strands can be synthesized in solution or on a solid surface by adding nucleotides in a defined manner and carrying out a cleavage reaction after each step. [Brief description of the drawings]

[0017] [Figure 1] 1 shows a nucleotide analogue with the 3'OH group capped with -CH2SSR. The base (B) can be a natural or unnatural nucleobase. The R group can be -Me, -Et, etc. The substituent X can be -H, -OH, or an -OH group capped with -Me, -Et, etc. [Diagram 2] FIG. 1 shows a preferred nucleotide structure where the capping group is —CH2SSMe. [Diagram 3] FIG. 1 shows the chemical structures of cleavage reagents such as DTT, DMPS, TCEP, and THPP. [Figure 4] Figure 1 shows the mechanism of cleavage of 3'-O-CH2SSMe protected nucleotides by phosphine or thiol compounds. It involves a two-step process. The first step is an instantaneous reaction, while the second step is a slow, rate-limiting step. Mg2+ can increase the rate of the second step hydrolysis reaction. [Diagram 5]FIG. 1 shows data plots for determining reaction constants. [Figure 6] FIG. 1 shows the half-lives of various cleavage reactions.

[0018] Detailed Description The present invention relates to the use of 3'-O-methylene alkyl disulfide blocked nucleotides (3'-OCH2SSR) in the enzymatic synthesis of DNA and RNA molecules. A key aspect of the present invention is that such nucleotides can be constructed in a variety of sizes depending on the structure of the R group. The smallest possible size among them is when R=Me. This is acceptable to polymerase enzymes used in DNA sequencing. It has been demonstrated that due to its small size, it fits into the active site of the polymerase and exhibits fast kinetics. An important aspect is that the capping group is stable yet easily removable in a fast chemical reaction of about 1.0 min under very mild DNA compatible conditions.

[0019] The present invention is based on compounds of general structure (I), which have been demonstrated to be excellent substrates for enzymatic incorporation when artificial polymerases such as Terminator 3, T9, etc. are used, and most importantly, they allow gentle removal of the capping (3'-OCH2SSR) without damaging the DNA molecule.

[0020] In one aspect, the nucleobases can be natural bases and modifications thereof (such as A, T, G, C, inosine, 5'-hydroxymethyl C and T), or non-natural nucleotides such as 7-deaza-G and 7-deaza-A and modifications thereof, where base B = non-natural base or base modification.

[0021] Preferably, the 3'-OH capping is a methylene alkyl disulfide (3'-O-CH2SSR), where the R group is methyl (R=-Me). Figures 1 and 2 show preferred nucleotides according to general formula (I).

[0022] X in formula (I) may be selected from the group consisting of hydrogen, hydroxyl, halogen, -OMe, -OEt, -OCH2SSMe, -OCH2CH2NH2.

[0023] The primer can be a nucleic acid initiator or seeding strand, such as a DNA, RNA or other polynucleotide strand. These can be single-stranded or double-stranded. Preferably, such a DNA or RNA strand can have a length of 1 to 50 nucleotides.

[0024] In another embodiment of the invention, the primers can be attached to a solid surface, such as a polymer surface, a particle, a microsphere or a nanosphere bead as known in microfluidic systems. Preferably, the primers are DNA or RNA strands attached to such a solid surface.

[0025] In another variation, the method can include a chemical treatment to remove the 3'-OH capping group with a cleavage reagent. Such a cleavage reagent can be selected from the group consisting of thiols, such as dithiolthreitol (DTT), dimercaptopropanesulfonic acid (DMPS), or phosphines, such as tris(2-carboxyethyl)phosphine (TCEP), tris(3-hydroxypropyl)phosphine (THPP). A preferred cleavage reagent is shown in FIG. 3.

[0026] Furthermore, cleavage can also be achieved by adding divalent ions such as Mg2+. In practice, salts such as MgSO4 or MgCl2 are added. Preferably, divalent ions are added at a pH range of 7-10 to facilitate the cleavage step in DNA and RNA synthesis in solution and in solid phase. Most preferred is the addition of dimercaptopropanesulfonic acid (DMPS) or other thiol compounds or phosphines as cleavage reagents, with or without Mg2+ salts. This embodiment is shown in the figures.

[0027] Working Example The general workflow for obtaining compounds according to formula (I) is shown in Scheme 1 below: [ka]

[0028] The synthesis of 3'-O-CH2SSMe capped nucleotides starts with the 3'-OCH2-S-trimethoxybenzyl analog (compound 1). This is triphosphorylated by standard methods such as the Ludwig or Eckstein method, and all base protecting groups are removed by NH4OH treatment, followed by lyophilization (or high vacuum pump) to remove volatile compounds and solvents. It is then resuspended in water, and the crude product is treated with DMTSF in acetate buffer at pH 4-6. The reaction is complete in 10 min. After neutralization with triethylamine and adjustment of the pH to 8.5, the crude product can be stored or immediately purified by ion exchange chromatography to obtain the desired product (compound 3) in about 25-40% yield. This method allows the production of MeSSCH2-capped reversible nucleotide terminators (MeSSdNTPs) in higher yields and can be fully scaled up to higher scales by linearly increasing the reagents and solvents using standard laboratory equipment, rapidly and significantly reducing the cost of nucleotide production.

[0029] As shown in Figure 4, the cleavage process occurs in two steps, the first step being the reductive cleavage of the disulfide bond followed by hydrolysis of the resulting thiol compound. The first step, reductive cleavage, occurs instantaneously, while the second step is slower and turns out to be the rate-limiting step. The second step is accelerated by the addition of salts (e.g., MgSO4, MgCl2).

[0030] 3'-O-(MeSSCH 2 Synthesis of 6-dGTP: The synthesis of 3'-O-CH2SSMe capped G nucleotides is shown below in Scheme 2: [ka]

[0031] In a 250 mL flask, 1.896 g (2.50 mmol) of 5'-OH-3'-O-(TMPMT)-dG(DPC)-Bu (4) was dried overnight under high vacuum in a desiccator in the presence of PO. It was dissolved by the addition of 4.0 mL of anhydrous pyridine and 7.0 mL of 1,4-dioxane. This mixture was treated with 2-chloro-1,3,2-benzodioxaphosphorin-4-one (0.6 g, 2.96 mmol). This mixture was stirred at room temperature for 15 minutes. A solution of tributylammonium pyrophosphate (1.8 g) was prepared by dissolving it in 5.0 mL of anhydrous DMF and 1.8 mL of BuN in a 50.0 mL centrifuge tube. This mixture was added in one portion to the vigorously stirred solution, and the resulting mixture was stirred at room temperature for 10 minutes. To this reaction mixture was added a solution of I2 (0.72 g, 2.84 mmol) prepared in 5.0 mL of pyridine and 0.5 mL of HPLC grade water. The resulting mixture was stirred for 15 min. The excess iodine was then quenched by the addition of 1.0 mL of 5% Na2SO3 in HPLC grade water. After stirring for 5 min, the reaction was diluted with 80 mL of HPLC grade water. The mixture was stirred at room temperature for 1.5 h. The resulting solution was decanted into a 1,000 mL round bottom flask, leaving behind a sticky reddish precipitate. The mixture was treated with 240 mL of 28-30% NH4OH and stirred at room temperature for 24 h. The mixture was concentrated to dryness by lyophilization. The mixture was resuspended in HPLC grade water and filtered to obtain 120 mL of crude filtrate of compound 5. The resulting crude product was diluted with 12 mL of 3.0 M acetate buffer at pH 5.2. To this vigorously stirred solution was added 1.6 gm of dimethyl(methylthio)sulfonium fluoroborate (DMTSF, 8.2 mmol) and the mixture was stirred at room temperature for about 10 minutes. The resulting cloudy mixture was neutralized with about 8.0 mL of Et3N and the precipitate was removed by centrifugation.The final product was obtained after ion exchange purification on a PL-SAX column (10μ, 1,000A, 50×150MM, Agilent, method: 0-5 min 100% A then 80% B over 50 min, flow rate 40mL / min; A=15% ACN / HO, B=15% ACN / 0.85M TEAB buffer) and then desalted by flash chromatography on a 275g C18Aq Teledyne column to give 691 μmol of compound 6 (27.6% yield) after lyophilization and resuspension in HPLC grade water (method: flow rate 80mL / min, A=100mM TEAB, B=ACN, equilibrated with 100% A for 15 min, gradient method: 0-5 min 100% A then 25% B over 5-45 min).

[0032] 3'-O-(MeSSCH 2 Synthesis of 3-dCTP (9): The synthesis of 3'-O-CH2SSMe capped C is shown below in Scheme 3: [ka]

[0033] 5'-OH-TMPMT-dC(Bz)7 (3.1 g, 5.6 mmol) was transferred to a 250 mL round bottom flask. The flask, along with a magnetic stir bar and a rubber septum, was dried overnight under high vacuum in a desiccator in the presence of P2O5. This was dissolved in anhydrous pyridine (8.4 mL) and anhydrous 1,4-dioxane (16.8 mL) and partially ground SalPCl solid (1.26 g, 6.2 mmol) was added. The reaction mixture was stirred at room temperature for 30 minutes. In a 50 mL centrifuge tube, a solution of tributylammonium pyrophosphate (3.8 g) in a mixture of Bu3N (3.9 mL) and anhydrous DMF (11.2 mL) was prepared. This was then added to the reaction mixture in one portion. The resulting reaction mixture was stirred at room temperature for 15 minutes. The product was oxidized with a solution of I2 (1.56 g, 6.2 mmol) in pyridine (11.2 mL) and H2O (1.1 mL, HPLC grade). The resulting brown mixture was stirred at room temperature for 20 min. The excess I2 was then quenched with 5% Na2SO3(aq.) (2.3 mL). The mixture was stirred at room temperature for 5 min. The mixture was further diluted with H2O (147 mL). It was stirred at room temperature for an additional 1.5 h. The reaction mixture was then treated with NH4-OH (28-30%, 110 mL). It was stirred at room temperature for 2 h and complete deprotection was confirmed by HPLC and LCMS. The reaction mixture was lyophilized and the residue was resuspended in HPLC grade water and filtered using a Corning filter system (0.22 μm). It was then transferred to a 250 mL bottle to give a solution of crude TMPMT dCTP (8) in H2O (~100 mL). The solution of TMPMT-dCTP (8) in the 250 mL bottle was further diluted with HO (90 mL) and then acetate buffer (3.0 M, pH 5.2, 20 mL) was added. To this mixture was immediately added DMTSF solid (2.9 g). The resulting reaction mixture was stirred at room temperature for 20 min. Complete conversion was confirmed by HPLC. The reaction mixture was neutralized to pH 7.5 with triethylamine (8.4 mL) and then filtered through a Corning™ disposable bottle-top filter system (250 mL, CA 0.45 μm).

[0034] The reaction mixture was purified by ion-exchange HPLC on a PL-SAX, 10 μm, 1000 Å, 50 × 150 mm (Agilent); method: 0-2 min 100% A, then 50 min 80% B, flow rate 40 mL / min, A=15% ACN / HO, B=15% ACN / 0.85 M TEAB buffer). The target peak was collected, pooled, and lyophilized. The residues were dissolved in HO (HPLC) and combined to give approximately 2.4 mmol of crude product 9 solution, approximately 89% pure by HPLC. The crude product was desalted by flash chromatography (Interchim PuriFlash- 450; 275 g RediSep Rf Gold HP C18 Aq column; method: 0-10 min 100% A, then 30 min 10% B; A=100 mM TEAB, B=ACN; flow rate 100 mL / min). The desired fractions were combined and lyophilized. The product was dissolved in 1X TE buffer and combined to give the desired pure MeSSdCTP (6) in 1X TE. A total of 90 mL (1,812 μmol, 36% from 9) was obtained with a purity of >99% by HPLC.

[0035] 3'-O-(MeSSCH 2 Synthesis of 12-dTTP: The synthesis of 3'-O-CH2SSMe capped T is shown in Scheme 4 below. [ka]

[0036] In a 250 mL round bottom flask, 4.7 g (10.06 mmol) of 5'-OH-3'-(TMPMT)-dT (10) was dried overnight under high vacuum in the presence of P2O5 as a drying agent. It was dissolved in 15.0 mL of anhydrous pyridine and 30.0 mL of anhydrous 1,4-dioxane under nitrogen atmosphere. This mixture was treated with 2-chloro-1,3,2-benzodioxaphosphorin-4-one (2.294 g, 11.2 mmol) under nitrogen atmosphere. This mixture was stirred at room temperature for 30 minutes. Then, in a 50.0 mL centrifuge tube, a solution of tributylammonium pyrophosphate (7.0 g) in 20.0 mL of anhydrous DMF and 7.0 mL of Bu3N was prepared. This mixture was added in one portion and stirred at room temperature for 10 minutes. The reaction product was oxidized with a solution of I2 (2.9 g, 11.4 mmol) in 20.0 mL pyridine and 2.0 mL HPLC grade water. The mixture was stirred for 15 min, resulting in a reddish solution. The excess iodine was then quenched by the addition of 4.0 mL 5% Na2SO3 in HPLC grade water and stirred for 5 min. The reaction mixture was transferred to a 1000 mL flask and diluted with 300 mL HPLC grade water. The clear solution was decanted into another 1000 mL, leaving a reddish brown precipitate. The mixture was first frozen in an acetonitrile-dry ice bath (-40 °C) and then frozen in a -75 °C freezer for approximately 1.0 h, resulting in lyophilization to give a thick paste-like product. The product is resuspended / extracted with HPLC grade water and the precipitate removed by filtration, the total volume at this point is approximately 480 mL (crude stock approximately 21 mM 3'-(TMPMT)-dTTP, 8, based on starting material 7).

[0037] Approximately half of the 480 mL of 3'-(TMPMT)-dTTP crude (11) (~240 mL, ~5.0 mmol) was taken into a separate plastic bottle. It was diluted with ~30 mL of 3.0 M acetate buffer at pH 5.2. It was treated with 3.0 g of DMTSF, and the mixture was stirred at room temperature for ~20 min. HPLC of the mixture showed complete conversion to MeSSdTTP. After removing the precipitate by centrifugation (using a 50 mL Falcon tube), the clear mixture (slightly yellowish) was taken into a separate flask. The mixture was adjusted to pH 7.0 by neutralizing with neat Et3N (~15 mL). At this point, the total volume of ~300 mL was divided into two portions of 150 mL (2.5 mmol) each. This was then run on a PL-SAX (10u, 1,000A, 50x150MM, Agilent), gradient: 0-5 min 100% A, then 5-50 min 80% B, flow rate: 40mL / min, A=15%ACN / HO, B=15%ACN / 0.85M TEAB buffer, R f Purification by ion exchange HPLC on the target peak at =23-28 min. Collected fractions were combined and lyophilized. Dried sample was resuspended in 100 mM TEAB buffer and desalted or final purified on a C18 Sunfire column. Yield: 780 micromoles, 31%, semi-pure. Final purification / desalting was performed on a C18 Sunfire Waters column (50X250MM): Method: 0-2 min 100% A, then 40% B over several minutes, flow rate 40 mL / min, A=100 mM, B=ACN. Yield 607 micromoles of compound 12 (overall yield approximately 24%).

[0038] 3'-O-(MeSSCH 2 Synthesis of 15-dATP: The synthesis of 3'-O-CH2SSMe capped A is shown below in Scheme 5: [ka]

[0039] In a 250 mL pear-shaped flask, 1.454 g (2.50 mmol) of 5'-OH-3'-(TMPMT)-dA-Bz (13) was dried overnight under high vacuum in the presence of PO. It was dissolved by the addition of 4.0 mL of anhydrous pyridine and 7.0 mL of anhydrous 1,4-dioxane under nitrogen atmosphere. The mixture was treated with 2-chloro-1,3,2-benzodioxaphosphorin-4-one (0.6 g, 2.96 mmol) and the mixture was stirred at room temperature for 15 min. In a 50.0 mL centrifuge tube, pyrophosphate Bu3NH in 5.0 mL of anhydrous DMF and 1.8 mL of Bu3N was dissolved. + A solution of I2 (1.8 g) was prepared. The mixture was added in one portion and stirred at room temperature for 10 min. The product was then oxidized by the addition of a solution of I2 (0.72 g, 2.84 mmol) dissolved in 5.0 mL of pyridine and 0.5 mL of HPLC grade water. The mixture was stirred for 15 min, resulting in a reddish solution. Excess iodine was quenched by the addition of 1.0 mL of 5% Na2SO3 in HPLC grade water and stirred for 5 min. The mixture was diluted with 80 mL of HPLC grade water and stirred at room temperature for 1.5 h. The color of the mixture changed to a clear solution, but a red sticky precipitate formed on the walls of the reaction flask. The clear solution was decanted into a new 1,000 mL round bottom flask, leaving a red precipitate. The mixture was treated with 240 mL of 28-30% NH4OH for 8 h. The product (14) was then lyophilized and dissolved in HPLC grade water to give a solution of about 100 mL (apparent concentration of about 25 mM based on the amount of starting material). This was diluted with 12 mL of 3.0 M acetate buffer at pH 5.2 and treated with 1.6 g of DMTSF for 20 min. The resulting cloudy mixture was neutralized with 6.0 mL of Et3N and centrifuged to remove the precipitate, resulting in a pH of about 9.0.

[0040] The product was purified by ion-exchange HPLC on a PL-SAX column (10u, 1,000A, 50x150MM, Agilent), HPLC gradient: 0-1 min 100% A, then 80% B over 50 min, flow rate: 40mL / min, A=15%ACN / HO, B=15%ACN / 0.85M TEAB buffer. The peak at Rf=25-30 min was processed for lyophilization. After lyophilization, the product was further purified by single injection on a 270gm C18AQ column (Teledyne), flow rate 80mL / min, A=100mM TEAB, B=ACN, equilibrated with 100% A for 15 min, 100% A for 0-5 min, then 25% B for 5-40 min. All fractions were collected and lyophilized. The product, MeSSdATP (15), was dissolved in 1× TE and the concentration was determined by UV spectrometry: total volume = 60 mL, C = 11.88 mM, yield = 712 μmol (28.5% yield), and purity >98%.

[0041] Examples of cleavage reactions: General Cleavage Kinetics Methodology The 3'-O capped nucleotide was treated with the cleavage agent mixture DMPS / THPP and the concentration of the 3' capped nucleotide was monitored at regular time intervals by UPLC with UV detector. From these data, the reaction rate constant k and reaction half-life t can be calculated according to the following equations as shown in Figure 5: ln[A]=-kt+ln[A] o , and t 1 / 2 =ln2 / k where [A] is the reactant concentration and k is the reaction rate constant.

[0042] experiment Nucleotides: MeSSdATP, 4.7 mM in 1x TE buffer; Cleavage agent mixture: 0, 5, 10, 20, 50, or 100 mM MgCl2 and 20 mM DMPS in pH 9.5 buffer.

[0043] 12.8 μL of nucleotide was added to 287 μL of cleavage agent mixture in a 1.5 mL UPLC sample vial. The resulting mixture was analyzed immediately by UPLC and then automatically every 10 min for up to 120 min. The area of ​​the initiating nucleotide peak was measured at each time point and used directly in the calculation. From the rate of decrease or disappearance of the intermediate peak area, the rate constant and half-life of the intermediate were calculated as described above. The results are shown in Figure 6 and show that the use of 10 mM Mg2+ in the cleavage agent mixture alone reduced the half-life by 30%, from 26.9 s to 18.3 s.

Claims

1. 1. A method for synthesizing a DNA or RNA strand, comprising: a) providing a primer capable of binding to a nucleotide; b) General formula (I) 【Chemistry 1】 [In the formula, B is a natural or unnatural nucleobase; R is -Me, -Et, -Pr, -iPr, -iBu, CH ２ CH ２ NH ２ or CH ２ CH ２ CN, R1 and R2 are each H, D, -Me, -Et, -Pr, -iPr, -iBu, or CHF ２ CH ２ CH ２ F, X is selected from the group consisting of hydrogen, hydroxyl, halogen, -OMe, -OEt, -OCH 2 SSMe and -OCH 2 CH 2 NH 2 ; c) providing a terminal transferase to ligate the first nucleotide to the primer; d) cleaving and removing the protecting group SS-R from the first nucleotide; e) repeating steps b) to d) by providing further nucleotides according to general formula (I) to obtain a DNA or RNA strand; A method comprising:

2. 2. The method of claim 1, wherein the primer is a DNA or RNA strand.

3. 3. The method according to claim 1, wherein the primer is a DNA or RNA strand attached to a solid surface.

4. 3. The method according to claim 1 or 2, characterized in that step c) is carried out by providing a cleavage reagent.

5. 5. The method of claim 4, wherein the cleavage reagent is selected from the group consisting of thiols, dithiolthreitol (DTT), dimercaptopropanesulfonic acid (DMPS), phosphines, tris(2-carboxyethyl)phosphine (TCEP), and tris(3-hydroxypropyl)phosphine (THPP).

6. 3. The method according to claim 1, wherein step c) is carried out by providing divalent ions.

7. 3. The method according to claim 1, wherein step c) is carried out at a pH in the range of 7 to 10.

8. 3. The method according to claim 1, wherein the first nucleotide and the further nucleotide provided in steps b) and e) according to general formula (I) do not comprise a detectable label.

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