Method for sequencing L-polynucleotides

Abstract

The present disclosure provides a novel method for amplifying and sequencing L-polynucleotides. Also provided are L-nucleotides and D-enzymes that can be used in the method. Methods for producing L-nucleotides and D-enzymes are also provided. The development of mirror-image biomolecules and systems faces a severe barrier due to the lack of efficient and reliable techniques for sequencing L-polynucleotides. The present disclosure provides a means to solve this problem.

Description

[Technical field]

[0001] 1. Sequence Listing This application contains a sequence listing. [Background technology]

[0002] 2.Background Chirality is a geometric property of a molecule. A molecule is chiral if it cannot be superimposed on its mirror image through a combination of rotation, translation, and some conformational change.

[0003] Many natural biomolecules, such as proteins, DNA, and RNA, are chiral. Amino acids other than glycine have a chiral carbon atom adjacent to the carboxyl group. This chiral center allows amino acids to exist as stereoisomers, also called enantiomers, which can be characterized as either L or D based on optical activity. However, most amino acids found in nature have the L form, except for glycine, which has no chirality. Nucleic acids have their chiral center on their backbone. Unlike amino acids, nucleic acids are predominantly D-form in nature. Due to chiral specificity, biomolecules can only interact and utilize other molecules or substrates if they have a particular chirality. For example, L-form polymerases can synthesize polynucleotides using D-form nucleotides, but cannot synthesize polynucleotides using L-form nucleotides.

[0004] In recent years, researchers have generated mirror-image forms of some biomolecules in an attempt to create mirror-image artificial systems based on D-amino acids, L-nucleic acids, and D-polymerases. In addition, mirror-image therapeutic proteins or polynucleotides have been developed, for example with advantageous slower biodegradation in the body. However, the development of mirror-image biomolecules and systems faces severe barriers due to the lack of efficient and reliable techniques for sequencing L-polynucleotides. Summary of the Invention [Means for solving the problem]

[0005] 3. Abstract The present disclosure provides a novel method for sequencing L-polynucleotides.Also provides compositions for sequencing methods, such as novel L-nucleotides and D-enzymes.It is expected that the sequencing method will enable high-throughput sequencing of L-polynucleotides.

[0006] Thus, in a first aspect, the disclosure provides a method for the preparation of a pentose sugar comprising: a. a pentose sugar selected from (3R,4S)-3,4,5-trihydroxypentanal and (4R)-4,5-dihydroxypentanal; wherein the H of the 5' hydroxyl is replaced by one or more phosphate groups; and the H of the 3' hydroxyl group, if present, is optionally replaced by a cleavable protecting group; and b. nitrogen bases; c. optionally, a cleavable label comprising a cleavable linker and a label; A mirror image nucleotide comprising: A mirror nucleotide is provided in which there is at least one cleavable protecting group and a cleavable label.

[0007] In certain embodiments, the cleavable label is linked to the nitrogenous base, the 3'O, or the 5' phosphate group.

[0008] In certain embodiments, the mirror nucleotide has Formula I: [ka] having a structure according to where the base is a nitrogenous base and R' is a cleavable protecting group.

[0009] In certain embodiments, the mirror nucleotide has the formula II [ka] having a structure according to where base is a nitrogenous base; R' is a cleavable protecting group or H; R2-label is a cleavable label comprising a cleavable linker R2 and a label.

[0010] In certain embodiments, the mirror nucleotide has formula III: [ka] having a structure according to where base is a nitrogenous base; R2-label is a cleavable label comprising a cleavable linker R2 and a label.

[0011] In certain embodiments of the mirror nucleotide of formula II or III, the label is: [ka] is selected from the group consisting of:

[0012] In certain embodiments of the mirror image nucleotides of Formula I or II, the cleavable protecting group is selected from allyl, dimethyl disulfide, nitrobenzyl, and azide protecting groups.

[0013] In certain embodiments of the mirror nucleotides of formula I or II, the cleavable protecting group is: [ka] is selected from.

[0014] In certain embodiments of the mirror image nucleotides of Formula II or III, the cleavable linker is photocleavable, cleaved by contact with a water-soluble phosphine, or cleaved by a water-soluble transition metal-containing catalyst.

[0015] In certain embodiments of the mirror image nucleotides of Formula II or III, the cleavable linker comprises an allyl group, a disulfide group, or an azide group.

[0016] In certain embodiments of the mirror image nucleotides of formula II or III, the cleavable linker is: [ka] Includes.

[0017] In certain embodiments of the mirror image nucleotide of formula I, the compound is: [ka] The structure is selected from:

[0018] In certain embodiments, R' comprises a methyl disulfide, allyl, azide, or nitrobenzyl moiety.

[0019] In certain embodiments, R' is: [ka] is selected from.

[0020] In certain embodiments of the mirror nucleotide of formula II, the compound is: [ka] [ka] The structure is selected from:

[0021] In certain embodiments, R' comprises a methyl disulfide, allyl, azide, or nitrobenzyl moiety.

[0022] In certain embodiments, R' is: [ka] is selected from.

[0023] In certain embodiments, R2 is: [ka] Includes.

[0024] In certain embodiments of the mirror nucleotide of formula III, the compound is: [ka] [ka] The structure is selected from:

[0025] In certain embodiments, R2 is: [ka] Includes.

[0026] In certain embodiments of the mirror nucleotide of formula II, the compound is: [ka] [ka] having a structure selected from R' is: [ka] is selected from.

[0027] In certain embodiments of the mirror nucleotide of formula III, the compound is: [ka] [ka] having a structure selected from:

[0028] In certain embodiments, R' comprises a methyl disulfide, allyl, azide, or nitrobenzyl moiety.

[0029] In certain embodiments, R' is: [ka] is selected from.

[0030] In a second aspect, the disclosure provides a mirror image nucleic acid polymerase comprising a sequence having at least 90% sequence identity to SEQ ID NO:1, wherein the polymerase comprises D-form amino acids.

[0031] In certain embodiments, the polymerase is composed of D-amino acids.

[0032] In certain embodiments, the polymerase comprises a sequence having at least 95% sequence identity to SEQ ID NO: 1. In further embodiments, the polymerase comprises a sequence having at least 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1.

[0033] In certain embodiments, the polymerase comprises one or more modifications at one or more amino acid sites selected from E276, K317, N424, and S651, compared to SEQ ID NO:1.

[0034] In certain embodiments, the polymerase comprises one or more substitutions selected from E276A, K317G, N424A, and S651A compared to SEQ ID NO:1.

[0035] In certain embodiments, the polymerase comprises the following substitutions compared to SEQ ID NO:1: E276A, K317G, N424A, and S651A.

[0036] In certain embodiments, the polymerase comprises one or more modifications at one or more amino acid sites selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733, and I744, compared to SEQ ID NO:1.

[0037] In certain embodiments, the polymerase comprises one or more substitutions of Ile to Ala, Val, Leu, or Tyr at one or more amino acid sites selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733, and I744, compared to SEQ ID NO:1.

[0038] In certain embodiments, the polymerase comprises one or more substitutions selected from I80V, I127V, I171A, I176V, I191V, I228V, I256V, I264A, I268L, I400V, I597V, I610V, I618A, I630L, I642V, I715Y, I733V, and I744V compared to SEQ ID NO:1.

[0039] In certain embodiments, the polymerase comprises the following substitutions compared to SEQ ID NO:1: I80V, I127V, I171A, I176V, I191V, I228V, I256V, I264A, I268L, I400V, I597V, I610V, I618A, I630L, I642V, I715Y, I733V, and I744V.

[0040] In certain embodiments, the polymerase comprises one or more modifications at one or more amino acid sites selected from M129, I130, G131, D141, E143, L408, Y409, P410, A485, T514, and I521, compared to SEQ ID NO:1.

[0041] In certain embodiments, the polymerase comprises one or more modifications at one or more amino acid sites selected from D141, E143, Y409, and A485, compared to SEQ ID NO:1.

[0042] In certain embodiments, the polymerase comprises one or more substitutions selected from D141A, E143A, Y409V, and A485L compared to SEQ ID NO:1.

[0043] In certain embodiments, the polymerase comprises the following substitutions compared to SEQ ID NO:1: D141A, E143A, Y409V, and A485L.

[0044] In certain embodiments, the polymerase comprises one or more modifications at one or more amino acid sites selected from D141, E143, L408, Y409, P410, A485, T514, and I521, compared to SEQ ID NO:1.

[0045] In certain embodiments, the polymerase comprises one or more substitutions selected from D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L compared to SEQ ID NO:1.

[0046] In certain embodiments, the polymerase comprises the following substitutions compared to SEQ ID NO:1: D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L.

[0047] In certain embodiments, the polymerase comprises one or more modifications selected from the following substitutions compared to SEQ ID NO:1: M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S, I521L, or the addition of a D between I130 and G131.

[0048] In certain embodiments, the polymerase comprises the following substitutions compared to SEQ ID NO:1: M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L, as well as the addition of a D between I130 and G131.

[0049] In certain embodiments, the polymerase comprises a sequence selected from SEQ ID NOs: 2-7.

[0050] In certain embodiments, the polymerase consists of a sequence selected from SEQ ID NOs: 2-7.

[0051] In a third aspect of the present disclosure, there is provided a method for replicating an L-polynucleotide, the method comprising the step of inducing replication of the L-polymerase by incubating a mixture containing (i) an L-polynucleotide, (ii) an L-primer, (iii) an L-dNTP, (iv) a polymerase according to any one of claims 1 to 38, and (v) a buffer.

[0052] In a particular embodiment the L-polynucleotide is DNA or RNA.

[0053] In certain embodiments, the mixture comprises L-dATP, L-dGTP, L-dCTP, and L-dTTP.

[0054] In a particular embodiment, the buffer comprises 50 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 1 mM DTT, and 50 mM KCl.

[0055] In certain embodiments, the incubation step comprises PCR.

[0056] In a fourth aspect of the present disclosure, there is provided a method for sequencing an L-polynucleotide comprising: a. Obtaining a replication product by incubating a mixture containing (i) an L-polynucleotide, (ii) an L-primer, (iii) an L-3'-O-R'-dNTP-R2-label, (iv) a polymerase according to any one of claims 17 to 38, and (v) a buffer; b. detecting a signal from the L-3'-O-R'-dNTP-R2-label incorporated into the replication product; and c. Induce cleavage of the R' and R2 groups of the L-3'-O-R'-dNTP-R2-label incorporated into the replication product A method is provided that includes a cycle.

[0057] In certain embodiments, the cycle is repeated at least 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 times.

[0058] In certain embodiments, the L-3'-O-R'-dNTP-R2-label comprises an L-3'-O-R'-dATP-R2-label, an L-3'-O-R'-dTTP-R2-label, an L-3'-O-R'-dGTP-R2-label, and an L-3'-O-R'-dCTP-R2-label, each label being different. In certain embodiments, the L-3'-O-R'-dNTP-R2-label has a structure according to Formula II as defined in Section 5.2.2.1.2. In certain embodiments, the signal is a fluorescent signal.

[0059] In a fifth embodiment of the present disclosure, there is provided a method for sequencing an L-polynucleotide comprising: a. obtaining a replication product by incubating a mixture containing (i) an L-polynucleotide, (ii) an L-primer, (iii) an L-dNTP, (iv) an L-ddNTP-R2-labeled or an L-3'-O-R'-dNTP-R2-labeled, (v) the polymerase according to any one of claims 17 to 38, and (vi) a buffer; b. separating the replication products; c. detecting a signal from the L-ddNTP-R2-label or L-3'-O-R'-dNTP-R2-label incorporated into the replication product; A method is provided, comprising:

[0060] In certain embodiments, L-dNTP comprises L-dATP, L-dTTP, L-dGTP and L-dCTP. In certain embodiments, L-ddNTP-R2-label comprises L-ddATP-R2-label, L-ddTTP-R2-label, L-ddGTP-R2-label and L-ddCTP-R2-label, each label being different.

[0061] In certain embodiments, the L-ddNTP-R2-label has formula III as defined in section 5.2.2.2.1. In certain embodiments, the L-3'-O-R'-dNTP-R2-label comprises an L-3'-O-R'-dATP-R2-label, an L-3'-O-R'-dTTP-R2-label, an L-3'-O-R'-dGTP-R2-label, and an L-3'-O-R'-dCTP-R2-label, each label being different. In certain embodiments, the L-3'-O-R'-dNTP-R2-label has formula II as defined in section 5.2.2.1.2. In these particular embodiments, the signal is a fluorescent signal.

[0062] In certain of these embodiments, the incubation step comprises PCR. In certain of these embodiments, the separation step comprises separating the replication products by size.

[0063] In a sixth embodiment of the present disclosure, there is provided a method for sequencing an L-polynucleotide comprising: a. Obtaining a replication product by incubating a mixture containing (i) an L-polynucleotide, (ii) an L-primer, (iii) an L-3'-O-R'-dNTP, (iv) an L-ddNTP-R2-labeled or an L-dNTP-R2-labeled, (v) a polymerase according to any one of claims 17 to 38, and (vi) a buffer; b. detecting a signal from an L-ddNTP-R2-label or an L-3'-O-R'-dNTP-R2-label incorporated into the replication product; and ci) the R' group of the L-3'-O-R'-dNTP and the R2 group of the L-ddNTP-R2-label that are incorporated into the replication product; or ii) inducing cleavage of the R' group of the L-3'-O-R'-dNTP, the R' and R2 groups of the L-3'-O-R'-dNTP-R2-label that are incorporated into the replication product. A method is provided that includes a cycle.

[0064] In certain embodiments, the cycle is repeated at least 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 times.

[0065] In certain embodiments, the L-3'-O-R'-dNTPs include L-3'-O-R'-dATP, L-3'-O-R'-dTTP, L-3'-O-R'-dGTP, and L-3'-O-R'-dCTP.

[0066] In certain embodiments, the L-3'-O-R'-dNTP has a structure according to Formula I, as defined in Section 5.2.2.1.1.

[0067] In certain embodiments, the L-ddNTP-R2-label comprises an L-ddATP-R2-label, an L-ddTTP-R2-label, an L-ddGTP-R2-label, and an L-ddCTP-R2-label, each label being different.

[0068] In certain embodiments, the L-ddNTP-R2-label has a structure according to formula III as defined in Section 5.2.2.2.1. In certain embodiments, the L-3'-O-R'-dNTP-R2-label includes an L-3'-O-R'-dATP-R2-label, an L-3'-O-R'-dTTP-R2-label, an L-3'-O-R'-dGTP-R2-label, and an L-3'-O-R'-dCTP-R2-label, each label being different. In certain embodiments, the L-3'-O-R'-dNTP-R2-label has a structure according to formula II as defined in Section 5.2.2.1.2. In certain embodiments, the signal is a fluorescent signal.

[0069] In a sixth aspect of the present disclosure, there is provided a kit for replicating an L-polynucleotide comprising (i) a polymerase according to any one of the preceding embodiments, and (ii) optionally a buffer.

[0070] In certain embodiments, the kit comprises L-dNTPs. In certain embodiments, the L-dNTPs comprise L-dATP, L-dGTP, L-dCTP, L-dTTP, or L-UTP.

[0071] In certain embodiments, the kit comprises an L-3'-O-R'-dNTP-R2-label. In certain embodiments, the kit comprises an L-3'-O-R'-dNTP-R2-label, an L-3'-O-R'-dATP-R2-label, an L-3'-O-R'-dTTP-R2-label, an L-3'-O-R'-dGTP-R2-label, an L-3'-O-R'-dCTP-R2-label, or an L-3'-O-R'-dUTP-R2-label. In certain embodiments, the kit comprises an L-3'-O-R'-dNTP-R2-label having Formula II as defined in Section 5.2.2.1.2.

[0072] In certain embodiments, the kit comprises an L-ddNTP-R2-label. In certain embodiments, the kit comprises an L-ddNTP-R2-label, including an L-ddATP-R2-label, an L-ddTTP-R2-label, an L-ddGTP-R2-label, an L-ddCTP-R2-label, or an L-ddUTP-R2-label. In certain embodiments, the L-ddNTP-R2-label has formula III as defined in section 5.2.2.2.1.

[0073] In certain embodiments, the kit comprises an L-3'-O-R'-dNTP. In certain embodiments, the L-3'-O-R'-dNTP comprises an L-3'-O-R'-dATP, an L-3'-O-R'-dTTP, an L-3'-O-R'-dGTP, and an L-3'-O-R'-dCTP. In certain embodiments, the L-3'-O-R'-dNTP has Formula I as defined in Section 5.2.2.1.1.

[0074] 4. Brief description of the drawings These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description and accompanying drawings. [Brief description of the drawings]

[0075] [Figure 1] Figure 1 provides the amino acid sequence of the (D)-type mutant 9°N DNA polymerase (Candidate #1-1) (SEQ ID NO: 8). All amino acids are D-type amino acids. Diamonds = mutations introduced for NCL; Circles = NCL sites (start of the "second" strand); Squares = potential substitution sites for isoleucine; Triangles = mutations introduced into modified nucleotides.

[0076] [Figure 2A] Figure 2A shows the sequence of the D-type 9°N DNA polymerase mutant 1-I, 9°NN fragment (SEQ ID NO: 9), with the sequences of the nine synthetic peptides presented on separate lines. Figure 2B shows the synthetic pathway of the D-type 9°N DNA polymerase mutant 1-I, 9°NN fragment. [Figure 2B] Same as above.

[0077] [Figure 3A] Figure 3A shows the sequence of D-type 9°N DNA polymerase mutant 1-I, 9°NC fragment (SEQ ID NO: 10), with the sequences of six synthetic peptides presented on separate lines. Figure 3B shows the synthetic pathway of D-type 9°N DNA polymerase mutant 1-I, 9°NC fragment. [Figure 3B] Same as above.

[0078] [Figure 4] Figure 4A provides the TLC staining of Tfa-(D)-Thz-OH, i.e. (S)-3-(2,2,2-trifluoroacetyl)thiazolidine-4-carboxylic acid. Figure 4B provides the H NMR (500 MHz, CDCl3) results of Tfa-(D)-Thz-OH, i.e. (S)-3-(2,2,2-trifluoroacetyl)thiazolidine-4-carboxylic acid.

[0079] [Diagram 5] FIG. 5 shows the synthesis of 2-Cl-(Trt)-NHNH2 as detailed in Example 13.

[0080] [Figure 6]FIG. 6A is the peptide sequence of pentamer D-9°oN-N-6 (SEQ ID NO: 11). FIG. 6B provides an analytical HPLC chromatogram (λ=214 nm) of pentamer crude D-9°NN-6. Column: Welch C4. Gradient: 0-20 min 100-80% A in B, 20-40 min 80-50% A in B, 40-60 min 50-20% A in B, and 60-70 min 20-0% A in B, where A=HO (0.1% TFA) and B=CH3CN (0.1% TFA). FIG. 6C provides an analytical HPLC chromatogram of pentamer pure D-9°NN-6. Gradient: 95-5% CH3CN (0.1% TFA) in HO (0.1% TFA) over 30 min. Purification conditions: Column: Welch C4 semi-preparative column. Gradient: 1-20 min 100-80% A in B, 20-40 min 80-50% A in B, 40-50 min 50-20% A in B, and 50-55 min 20-0% A in B, where A=HO (0.1% TFA) and B=CHCN (0.1% TFA).

[0081] [Figure 7] Figure 7A is the peptide sequence of the 11-mer D-9°N-N-6 (SEQ ID NO: 12). Figure 7B provides an analytical HPLC chromatogram (λ=214 nm) of the 11-mer crude D-9°oN-N-6. Column: Welch C4. Gradient: 95-5% CH3CN (0.1% TFA) in H2O (0.1% TFA) over 30 min. Figure 7C provides an analytical HPLC chromatogram of the 11-mer pure D-Pfu-9°oN-6. Gradient: 95-5% CH3CN (0.1% TFA) in H2O (0.1% TFA) over 30 min. Purification conditions: Column: Welch C4 semi-preparative column. Gradient: 1–20 min 100–80% A in B, 20–40 min 80–50% A in B, 40–50 min 50–20% A in B, and 50–55 min 20–0% A in B, where A = HO (0.1% TFA) and B = CHCN (0.1% TFA). Figure 7D provides the MALDI-TOF mass spectrum of the 11-mer D-9°oN-N-6.

[0082] [Figure 8]Figure 8A is the peptide sequence of the 24-mer D-9°oN-N-6 (SEQ ID NO: 13). Figure 8B provides an analytical HPLC chromatogram (λ=214 nm) of the 24-mer crude D-9°oN-N-6. Column: Welch C4. Gradient: 0-20 min 100-80% A in B, 20-40 min 80-50% A in B, 40-60 min 50-20% A in B, and 60-70 min 20-0% A in B, where A=HO (0.1% TFA) and B=CH3CN (0.1% TFA). Figure 8C provides an analytical HPLC chromatogram of the pentamer pure D-Pfu-9°oN-6. Gradient: 95-5% CH3CN (0.1% TFA) in HO (0.1% TFA) over 30 min. Purification conditions: Column: Welch C4 semi-preparative column. Gradient: 1-20 min 100-80% A in B, 20-40 min 80-50% A in B, 40-50 min 50-20% A in B, and 50-55 min 20-0% A in B, where A=H2O (0.1% TFA) and B=CH3CN (0.1% TFA).

[0083] [Figure 9] Figure 9A is the peptide sequence of the 9-mer D-9°NC-3 (SEQ ID NO: 14). Figure 9B provides an analytical HPLC chromatogram (λ=214 nm) of the 9-mer crude D-9°NC-3. Column: Welch C4. Gradient: 0-20 min 100-80% A in B, 20-40 min 80-50% A in B, 40-60 min 50-20% A in B, and 60-70 min 20-0% A in B, where A=H2O (0.1% TFA) and B=CH3CN (0.1% TFA). Purification conditions: Column: Polaris C18-A semi-preparative column. Gradient: 1–20 min 100–80% A in B, 20–40 min 80–50% A in B, 40–60 min 50–20% A in B, and 60–70 min 20–0% A in B, where A = HO (0.1% TFA) and B = CHCN (0.1% TFA). Figure 9C provides the HRMS (ESI) spectrum of the nonamer D-9°NC-3: [M+H] calcd for CHNOS 946.4053; found 946.4064.

[0084] [Figure 10] FIG. 10A provides an analytical HPLC chromatogram (λ=214 nm) of purified 33-mer D-9°NC-1. Column: Welch C4. Gradient: 0-20 min 75-35% A in B, 20-23 min 35-0% A in B, where A=H2O (0.1% TFA) and B=CH3CN (0.1% TFA). b) ESI-MS spectrum of 33-mer D-9°NC-1: Observed 3985.5, calculated 3985.6. FIG. 10B provides an ESI-MS spectrum of 33-mer D-9°NC-1.

[0085] [Figure 11] FIG. 11A provides an analytical HPLC chromatogram (λ=214 nm) of the 39-mer purified D-9oN-C-2. Column: Welch C4. Gradient: 0-20 min 75-35% A in B, 20-23 min 35-0% A in B, where A=H2O (0.1% TFA) and B=CH3CN (0.1% TFA). FIG. 11B provides an ESI-MS spectrum of the 39-mer D-9oN-C-2: observed 4921.5, calculated 4921.6.

[0086] [Figure 12] FIG. 12A provides the peptide sequence of the 21-mer D-9oN-C-3 (SEQ ID NO: 15). FIG. 12B provides an analytical HPLC chromatogram (λ=214 nm) of the 21-mer crude D-9oN-C-3. Column: Welch C4. Gradient: 0-30 min 90-50% A in B, 30-40 min 50-5% A in B, where A=H2O (0.1% TFA) and B=CH3CN (0.1% TFA). Purification conditions: Column: Polaris C18-A semi-preparative column. Gradient: 0-30 min 90-50% A in B, 30-40 min 50-5% A in B, where A=H2O (0.1% TFA) and B=CH3CN (0.1% TFA). FIG. 12C provides an ESI-MS spectrum of the 21-mer D-9°NC-3. FIG. 12D provides the MS spectrum of the 21-mer D-9° NC-3 without TFA protection and the observed mass of 2196.1.

[0087] [Figure 13] FIG. 13A is the peptide sequence of the Cys35-mer D-9oN-C-3 (SEQ ID NO: 16). FIG. 13B provides an analytical HPLC chromatogram (λ=214 nm) of the crude Cys35-mer D-9oN-C-3. Column: Welch C4. Gradient: 0-30 min 70-20% A in B, 30-40 min 20-5% A in B, where A=H2O (0.1% TFA) and B=CH3CN (0.1% TFA). Purification conditions: Column: Polaris C18-A semi-preparative column. Gradient: 0-30 min 70-20% A in B, 30-40 min 20-5% A in B, where A=H2O (0.1% TFA) and B=CH3CN (0.1% TFA). Figure 13C provides the ESI-MS spectrum of the 21-mer D-9°NC-3, and Figure 13D provides the observed mass of the Cys35-mer D-9°NC-3, 4276.

[0088] [Figure 14] FIG. 14A is the peptide sequence of the 56-mer D-9°NC-3 (SEQ ID NO: 17). FIG. 14B provides an analytical HPLC chromatogram (λ=214 nm) of the 56-mer crude D-9°NC-3. Column: Welch C4. Gradient: 0-30 min 80-30% A in B, 30-40 min 30-5% A in B, where A=H2O (0.1% TFA) and B=CH3CN (0.1% TFA). Purification conditions: Column: Polaris C18-A semi-preparative column. Gradient: 0-30 min 80-30% A in B, 30-40 min 30-5% A in B, where A=H2O (0.1% TFA) and B=CH3CN (0.1% TFA).

[0089] [Figure 15]FIG. 15A is the peptide sequence of the 35-mer D-Pfu-9oN-C-3 (SEQ ID NO: 18). FIG. 15B provides an analytical HPLC chromatogram (λ=214 nm) of the 35-mer crude D-Pfu-9oN-C-3. Column: Welch C4. Gradient: 0-30 min 80-30% A in B, 30-40 min 30-5% A in B, where A=HO (0.1% TFA) and B=CH3CN (0.1% TFA). Purification conditions: Column: Polaris C18-A semi-preparative column. Gradient: 0-30 min 80-30% A in B, 30-40 min 30-5% A in B, where A=HO (0.1% TFA) and B=CH3CN (0.1% TFA).

[0090] [Figure 16] FIG. 16A is the peptide sequence of 5-mer D-9oN-N-6 and 11-mer D-9oN-N-6 (SEQ ID NO: 19 and SEQ ID NO: 20). FIG. 16B provides a synthetic route for the preparation of 16-mer D-9oN-N-6. 5-mer D-9oN-N-6 (4 mg, 1 eq.) was dissolved in 0.4 mL of acidified ligation buffer (aqueous solution of 6 M Gn·HCl and 0.1 M NaH2PO4, pH 3.0). The mixture was cooled in an ice-salt bath (−15° C.) and 40 μl of 0.5 M NaNO2 (in acidified ligation buffer) was added. The reaction was kept in the ice-salt bath with stirring for 15 minutes, after which 0.4 ml of 0.2 M MPAA (in 6 M Gn·HCl and 0.1 M Na2HPO4, pH 5.7) was added. After addition of the 11-mer D-Pfu-9oN-N-6 (7.5 mg), the pH of the reaction mixture was adjusted to 6.6-6.8 with NaOH solution at room temperature. After 12 h, the reaction mixture was reduced with TCEP and purified by HPLC (purification conditions: 5-95% CH3CN (with 0.1% TFA) gradient in H2O (with 0.1% TFA) over 30 min on a Welch C4 column). The ligation product was obtained in 80% yield (9.0 mg). Figure 16C provides an analytical HPLC chromatogram (λ = 214 nm) of the peak conversion during the progression of the NCL reaction. Column: Welch C4. Gradient: 5-95% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) over 30 min.

[0091] [Figure 17] Figure 17A shows the peptide sequences of D-9oN-C-1 and D-9oN-C-2 (SEQ ID NOs: 21, 22, 23). Figure 17B provides a synthetic route for the preparation of the 72-mer D-9oN-C-7. The 33-mer D-9oN-C-1 (5.4 mg) was dissolved in 0.27 mL of acidified ligation buffer (aqueous solution of 6 M Gn·HCl and 0.1 M NaH2PO4, pH 3.0). The mixture was cooled in an ice-salt bath (-15°C) and 27 μl of 0.5 M NaNO2 (in acidified ligation buffer) was added. The reaction was kept in the ice-salt bath with stirring for 20 minutes, after which 0.14 ml of 0.4 M MPAA (in 8 M Gn·HCl and 0.1 M Na2HPO4, pH 5.7) was added. After addition of 39-mer D-9oN-C-2 (5.8 mg), the pH of the reaction mixture was adjusted to 6.6-6.8 with NaOH solution at room temperature. After 16 h, the mixture was purified by HPLC (purification conditions: 20-70% CH3CN (with 0.1% TFA) gradient in H2O (with 0.1% TFA) over 30 min on a Polaris C18 column). The ligation product was obtained. Figure 17C is an analytical HPLC chromatogram (λ=214 nm) of the peak conversion during the progression of the NCL reaction. Column: Welch C4. Gradient: 20-70% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) over 30 min. Figure 17D is an ESI-MS of 72-mer D-9°NC-7 with MPAA attached. FIG. 17E shows the ligation product with MPAA attached (observed mass: 9042; calculated mass: 9042). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0092] 5. Detailed Description 5.1.Definition "L-nucleic acid" or "L-nucleotide" refers to any nucleic acid or nucleotide molecule with L chirality, including but not limited to L-DNA, L-RNA, and hybrids thereof. The nucleic acid bases forming nucleic acid molecules can be bases A, C, G, T, and U, and derivatives thereof. Derivatives of these bases are well known in the art and are exemplified in PCR Systems, Reagents and Consumables (Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, New Jersey, USA).

[0093] The term "D-polymerase" as used herein refers to a protein that has the mirror form of an original polymerase with L-chirality. The original polymerase can be a naturally occurring or modified or synthetically produced polymerase. The original polymerase can be a DNA or RNA polymerase.

[0094] Although bases are commonly referred to as purines or pyrimidines, one of skill in the art will appreciate that derivatives and analogs are available that do not alter the ability of a nucleotide or nucleoside to undergo Watson-Crick base pairing. As used herein, the term "base" includes compounds or molecules whose core structure is the same as or closely resembles a natural base, but which have chemical or physical modifications, such as different or additional side groups, that allow the derivative nucleotide or nucleoside to be linked to another molecule. For example, a base can be a deazapurine.

[0095] "Hybridize" refers to the annealing of one single-stranded nucleic acid to another nucleic acid based on sequence complementarity. The tendency of hybridization between nucleic acids depends on the temperature and ionic strength of their environment, the length of the nucleic acid, and the degree of complementarity. The effect of these parameters on hybridization is well known in the art (see Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York).

[0096] Dehybridization is understood by those skilled in the art to mean dissociating hybridized primer (or its extended strand) from target nucleic acid without destroying target nucleic acid, thereby allowing further hybridization of second primer to target nucleic acid.In one embodiment, hybridization as used herein means stringent hybridization, for example, as described in Sambrook, J., Russell, DW, (2000) Molecular Cloning: A Laboratory Manual: 3rd Edition.

[0097] As used herein, hybridization of a primer sequence is intended to mean sufficient annealing such that the primer is extendable by creation of a phosphodiester bond.

[0098] Where a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit (where appropriate), between the upper and lower limits of that range, unless the context clearly dictates otherwise, is encompassed within the invention, as well as every other stated or intervening value within that stated range. The upper and lower limits of these smaller ranges may independently be included within the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0099] 5.2. Mirror Nucleotides Used in Sequencing 5.2.1. L-Nucleotides and L-Nucleosides In one embodiment, the disclosure provides a mirror image nucleoside or nucleotide modified to include at least one (i) cleavable protecting group on the 3'O of the pentose ring (if present) and (ii) a cleavable label. The mirror image nucleoside or nucleotide can be used in sequencing reactions, polynucleotide synthesis, nucleic acid amplification, nucleic acid hybridization assays, single nucleotide polymorphism studies, techniques that use enzymes such as polymerases, reverse transcriptases, terminal transferases, techniques that use labeled dNTPs (e.g., nick translation, random primer labeling, end labeling (e.g., with terminal deoxynucleotidyl transferase)), reverse transcription, or nucleic acid amplification.

[0100] In embodiments of the invention, chemically modified mirror image nucleosides or nucleotides are provided that include at least one (i) cleavable protecting group on the 3'O of the pentose sugar, if present, and (ii) a cleavable label. In embodiments where the mirror image nucleotide includes a cleavable label, the label is not particularly limited, so long as the label provides a means for detection and R' includes H.

[0101] 5.2.1.1 Bases In certain embodiments, the nitrogenous base is a purine or a pyrimidine. In certain embodiments, the nitrogenous base is a deazapurine. In certain embodiments, the nitrogenous base is thymine (5-methyl-2,4-dioxypyrimidine), cytosine (2-oxo-4-aminopyrimidine), 5-methyl-cytosine (2-oxo-5-methyl-4-aminopyrimidine), and uracil (2,4-dioxopyrimidine), adenine (6-aminopurine), 7-deazaadenine (7H-pyrrolo[2,3-d]pyrimidin-4-amine; 6-aminopurine). 2-amino-7-deazapurine), guanine (2-amino-6-oxypurine), 7-deazaguanine (2-amino-4-hydroxy-pyrrolo-[2,3-d]-pyrimidine; 2-amino-7-deaza-6-oxypurine), hypoxanthine (1,9-dihydro-6H-purin-6-one), xanthine (3,7-dihydro-1H-purine-2,6-dione), and 5-nitroindole. In certain embodiments, the nitrogenous base is thymine, uracil, cytosine, adenine, guanine, 7-deazaadenine, or 7-deazaguanine.

[0102] 5.2.1.2 R'-Cleavable Protecting Groups In certain embodiments, R' is selected from H and a cleavable protecting group. The cleavable protecting group of the present invention is not particularly limited, so long as the resulting nucleotide is an efficient substrate for the mirror image DNA polymerase.

[0103] Those skilled in the art will understand how to attach a suitable protecting group to the pentose ring to block interactions with the 3'-OH. The protecting group can be attached directly to the 3' position or to the 2' position (where the protecting group has sufficient size or charge to block interactions at the 3' position). Alternatively, the protecting group can be attached to both the 3' and 2' positions and cleaved to expose the 3'OH group.

[0104] Suitable protecting groups will be apparent to those skilled in the art and may be formed from any suitable protecting group disclosed in Greene & Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons. The protecting group must be removable (or modifiable) to generate a 3'OH group. The process used to obtain the 3'OH group may be any suitable chemical or enzymatic reaction.

[0105] In some embodiments, the cleavable protecting group comprises an allyl, nitrobenzyl, 1-methyl-2-alkyldisulfide, or methylazide moiety.

[0106] In certain embodiments, the cleavable protecting group is: [ka] is selected from where the wavy line demarks the point of attachment to the 3'O.

[0107] 5.2.1.3 Cleavable Label Constructs (R2-Labels) 5.2.1.3.1 Signs The present invention can use any conventional detectable label that can be modified to be covalently attached to the nucleotide via a cleavable linker.

[0108] Detection can be carried out by any suitable method, including fluorescence spectroscopy or other optical means. Particularly contemplated labels are fluorophores that emit radiation at a defined wavelength after absorbing energy. Many suitable fluorescent labels are known. For example, Welch et al. (Chem.Eur.J.5(3):951-960,1999) disclose dansyl-functionalized fluorescent moieties that can be used in the present invention. Zhu et al. (Cytometry 28:206-211,1997) describe the use of fluorescent labels Cy3 and Cy5, which can also be used in the present invention. Suitable labels for use are also disclosed by Prober et al. (Science 238:336-341, 1987); Connell et al. (BioTechniques 5(4):342-384, 1987); Ansorge et al. (Nucl. Acids Res. 15(11):4593-4602, 1987); and Smith et al. (Nature 321:674, 1986). Other commercially available labels include, but are not limited to, ATTO dyes (e.g., Atto 655 and Atto 647N), Quasar dyes, CF dyes, fluoresceins, rhodamines (including TMR, texas red, and Rox), Alexa Fluor® 647, 488, 532, 594, 633, Dyomics dyes, Bodipy (bodidpy), acridine, R6G, Cy3, Cy3.5, Cy5, Cy5.5, coumarin, pyrene, benzanthracene, and cyanines.

[0109] Although fluorescent labels are specifically contemplated, it will be clear to those skilled in the art that other forms of detectable labels are useful.For example, microparticles, including quantum dots (Empodocles, et al., Nature 399:126-130, 1999), gold nanoparticles (Reichert et al., Anal. Chem. 72:6025-6029, 2000), microbeads (Lacoste et al., Proc. Natl. Acad. Sci USA 97(17):9461-9466, 2000), and tags that can be detected by pH and voltage changes, mass spectrometry, Raman spectroscopy, and spectroscopic methods such as surface plasmon resonance (SPR) sensing can all be used.

[0110] i. Fluorophores In certain embodiments of the L-nucleotides of the present disclosure, the label is a fluorophore selected from Rox, bodipy, bodipy-FL-510, R6G, and Cy5, and functional derivatives thereof.

[0111] In certain embodiments, the fluorophore is: [ka] is selected from.

[0112] 5.2.1.3.2 Cleavable Linker (R2) Certain embodiments of the L-nucleotide of the present disclosure include a cleavable label linked, for example, to a nitrogenous base, a 3'O, or a 5' phosphate group via a cleavable linker. In certain embodiments, the linker is attached to a nitrogenous base. In certain embodiments, the linker is attached to the C8 of a purine base, the C7 of a 7-deazapurine base, or the C5 of a pyrimidine base.

[0113] In certain embodiments, the cleavable linker is photocleavable, cleaved by contact with a water-soluble phosphine, or cleaved by a water-soluble transition metal-containing catalyst.

[0114] Suitable linkers known to those of skill in the art include, but are not limited to, disulfide linkers, acid labile linkers (including dialkoxybenzyl linkers, Sieber linkers, indole linkers, t-butyl Sieber linkers), electrophilically cleavable linkers, nucleophilically cleavable linkers, photocleavable linkers, cleavage under reducing conditions, oxidative conditions, cleavage through the use of a safety catch linker, and cleavage by an excision mechanism.

[0115] i. Electrophilically cleavable linkers. Electrophilically cleaved linkers are typically cleaved by protons, including acid-sensitive cleavage. Suitable linkers include modified benzyl systems such as trityl, p-alkoxybenzyl esters, and p-alkoxybenzyl amides. Other suitable linkers include tert-butyloxycarbonyl (Boc) groups and acetal systems.

[0116] The use of electrophilic metals such as nickel, silver, or mercury in the cleavage of thioacetals or other sulfur-containing protecting groups can also be considered for the preparation of suitable linker molecules.

[0117] ii. A nucleophilically cleavable linker. Nucleophilic cleavage is also a well-recognized method in the preparation of linker molecules. Groups such as esters that are unstable in water (i.e., can be easily cleaved at basic pH) and groups that are labile to non-aqueous nucleophiles can be used. Fluoride ions can be used to cleave silicon-oxygen bonds in groups such as triisopropylsilane (TIPS) or t-butyldimethylsilane (TBDMS).

[0118] iii. Photocleavable linkers. Photocleavable linkers are widely used in carbohydrate chemistry. The light required to activate cleavage should preferably not affect other components of the modified nucleotide. For example, if a fluorophore is used as a label, it should preferably absorb light at a wavelength different from the wavelength required to cleave the linker molecule. Suitable linkers include linkers based on O-nitrobenzyl and nitroveratryl compounds. Benzoin chemistry-based linkers can also be used (Lee et al., J.Org.Chem.64:3454-3460,1999).

[0119] iv. Cleavage under reducing conditions Many linkers are known that are susceptible to reductive cleavage. Catalytic hydrogenation using palladium-based catalysts has been used to cleave benzyl and benzyloxycarbonyl groups. Reduction of disulfide bonds is also known in the art.

[0120] v. Cleavage under oxidizing conditions Oxidation-based approaches are well known in the art. Oxidation-based approaches include the oxidation of p-alkoxybenzyl groups and the oxidation of sulfur and selenium linkers. The use of water-soluble iodine to cleave disulfides and other sulfur or selenium-based linkers is also within the scope of the present invention.

[0121] vi. Safety catch linker Safety catch linkers are linkers that cleave in two steps. In a preferred system, the first step is the generation of a reactive nucleophilic center, followed by a second step involving intramolecular cyclization that results in cleavage. For example, a levulinic acid ester bond can be treated with hydrazine or photochemistry to release an active amine, which can then be cyclized to cleave the ester elsewhere in the molecule (Burgess et al., J. Org. Chem. 62:5165-5168, 1997).

[0122] vii. Cleavage by removal mechanism Elimination reactions can also be used, for example base catalyzed removal of groups such as Fmoc and cyanoethyl, and palladium catalyzed reductive removal of allylic systems.

[0123] 5.2.1.3.3 Spacers Similar to the cleavage site, the linker may contain one or more spacer moieties. The spacer (linker and bridge) may be, for example, any spacer typically used in the synthesis of bioconjugates. The spacer distances the nucleotide base from the cleavage site or the label. The length of the linker is not important, as long as the label is held at a sufficient distance from the nucleotide so as not to interfere with any interaction between the nucleotide and the enzyme.

[0124] In certain embodiments, the cleavable linker comprises an allyl group, a disulfide group, or an azide group along with an additional spacer. [ka] Includes.

[0125] 5.2.1.3.4 Cleavable Label Constructs In certain embodiments of the mirror image nucleosides or nucleotides provided herein, the cleavable label comprises a label described in Section 5.2.1.3.1 and a cleavable linker in more detail in Section 5.2.1.3.2.

[0126] In certain embodiments, the cleavable label construct comprises: [ka] Selected from; In the formula, the wavy lines demark the points of attachment to the mirror nucleotide, including attachment to a linker / spacer that is attached to the nucleotide to allow for the attachment of, for example, an amine moiety.

[0127] 5.2.2. Embodiments of the Disclosure In certain embodiments, pentose sugars selected from (3R,4S)-3,4,5-trihydroxypentanal and (4R)-4,5-dihydroxypentanal; (C-5 of the sugar is replaced by a hydroxyl, monophosphate, diphosphate, or triphosphate; C-3 of the sugar is substituted by H or O-R', where R' is H or a cleavable protecting group; Nitrogen bases; and Optionally, a cleavable label An L-nucleoside or L-nucleotide is provided, comprising: The L-nucleoside or L-nucleotide comprises at least one selected from (i) a cleavable protecting group and (ii) a cleavable label.

[0128] The embodiments provided herein are further described with reference to nucleotides, however, unless otherwise indicated, reference to a nucleotide is intended to be applicable to a nucleoside as well.

[0129] In certain embodiments, the compound of formula A [ka] L-nucleotides are provided having a structure according to wherein R' is H or a cleavable protecting group; X is H or hydroxyl, the base is a nitrogenous base, the nucleotide optionally comprises a cleavable label, and at least one cleavable protecting group or cleavable label is present.

[0130] In certain embodiments, the nitrogenous base is as described herein in Section 5.2.1.1. In certain embodiments, the cleavable protecting group is as described herein in Section 5.2.1.2. In certain embodiments, the cleavable label is as described herein in Section 5.2.1.3.

[0131] In any of the described embodiments, the location of the cleavable label is not particularly limited and may be linked to the nucleotide through any position that is chemically feasible and / or generally known in the art. In certain embodiments, the cleavable label is attached to the nitrogenous base via the terminal phosphate or at the 3'O, in which case the cleavable protecting group is the cleavable label.

[0132] 5.2.2.1(L) Nucleotide Reversible Terminators (L-NRTs) 5.2.2.1.1 [(L)-3'-O-R'-dNTP]-Formula I In one embodiment of the present disclosure, a compound of formula I: [ka] L-nucleotides are provided having a structure according to where the base is a nitrogenous base and R' is a cleavable protecting group.

[0133] In certain embodiments, the nitrogenous base is as described herein in Section 5.2.1.1 In certain embodiments, the cleavable protecting group is as described herein in Section 5.2.1.2.

[0134] In certain embodiments: [ka] Provided is an L-nucleotide having a structure according to:

[0135] In certain embodiments, R' comprises a cleavable protecting group as described herein in Section 5.2.1.2. In further embodiments, the cleavable protecting group R' is: [ka] is selected from where the wavy line demarks the point of attachment to the 3'O.

[0136] 5.2.2.1.2 [(L)-3'-O-R'-dNTP-R2-label]-Formula II In one embodiment of the present disclosure, a compound of formula II: [ka] L-nucleotides are provided having a structure according to where base is a nitrogenous base; R' is a cleavable protecting group or H; and label-R2 together comprise a cleavable label construct.

[0137] In certain embodiments, the nitrogenous base is as described herein in Section 5.2.1.1. In certain embodiments, the cleavable protecting group is as described herein in Section 5.2.1.2. In certain embodiments, the cleavable label construct is as described herein in Section 5.2.1.3.

[0138] In certain embodiments: [ka] [ka] Provided is an L-nucleotide having a structure selected from:

[0139] In certain embodiments, the cleavable protecting group R' is as described herein in Section 5.2.1.2. In certain embodiments, the cleavable linker R2 is as described herein in Section 5.2.1.3.2.

[0140] In certain embodiments, R' is: [ka] is selected from where the wavy line demarks the point of attachment to the 3'O.

[0141] In certain embodiments, R2 is: [ka] Includes.

[0142] In certain embodiments: [ka] L-nucleotides having a structure selected from wherein R' is H or the cleavable protecting group is as described in Section 5.2.1.2 herein.

[0143] In certain embodiments, R' is: [ka] is selected from where the wavy line demarks the point of attachment to the 3'O.

[0144] 5.2.2.2 Labeled Terminators 5.2.2.2.1 [(L)-ddNTP-R2-label] Formula III In one embodiment of the present disclosure, a compound of formula III: [ka] L-nucleotides are provided having a structure according to where Base is a nitrogenous base; R2 is a cleavable linker; and Label-R2 together comprise a cleavable label construct.

[0145] In certain embodiments, the nitrogenous base is as described herein in Section 5.2.1.1. In certain embodiments, the cleavable label construct, Label-R2, is as described herein in Section 5.2.1.3.

[0146] In certain embodiments: [ka] [ka] Provided is an L-nucleotide having a structure selected from:

[0147] In certain embodiments, the cleavable linker R2 is as described in Section 5.2.1.3.2 herein.

[0148] In certain embodiments, R2 is: [ka] Includes.

[0149] In certain embodiments: [ka] [ka] Provided is an L-nucleotide having a structure selected from:

[0150] 5.3. Mirror-Image Polymerase In another aspect, the present disclosure provides a mirror polymerase. Specifically, the mirror polymerase comprises D-amino acids. In some embodiments, the mirror polymerase consists of D-amino acids. In some embodiments, the mirror polymerase comprises both D-amino acids and L-amino acids. In some embodiments, the mirror polymerase does not comprise L-amino acids.

[0151] In some embodiments, the mirror image nucleic acid is a mirror image of a DNA polymerase or an RNA polymerase. In some embodiments, the mirror image nucleic acid is a mirror image of a 9°N DNA polymerase or a modification thereof.

[0152] In some embodiments, the mirror polymerase comprises a sequence having at least 90% sequence identity to SEQ ID NO:1. In some embodiments, the mirror polymerase comprises a sequence having at least 96% sequence identity to SEQ ID NO:1. In some embodiments, the mirror polymerase comprises a sequence having at least 96% sequence identity to SEQ ID NO:1. In some embodiments, the mirror polymerase comprises a sequence having at least 97% sequence identity to SEQ ID NO:1. In some embodiments, the mirror polymerase comprises a sequence having at least 98% sequence identity to SEQ ID NO:1. In some embodiments, the mirror polymerase comprises a sequence having at least 99% sequence identity to SEQ ID NO:1. In some embodiments, the mirror polymerase comprises the sequence of SEQ ID NO:1.

[0153] In some embodiments, the mirror polymerase has a sequence having at least 90% sequence identity to SEQ ID NO:1. In some embodiments, the mirror polymerase has a sequence having at least 96% sequence identity to SEQ ID NO:1. In some embodiments, the mirror polymerase has a sequence having at least 96% sequence identity to SEQ ID NO:1. In some embodiments, the mirror polymerase has a sequence having at least 97% sequence identity to SEQ ID NO:1. In some embodiments, the mirror polymerase has a sequence having at least 98% sequence identity to SEQ ID NO:1. In some embodiments, the mirror polymerase has a sequence having at least 99% sequence identity to SEQ ID NO:1. In some embodiments, the mirror polymerase has the sequence of SEQ ID NO:1.

[0154] In some embodiments, the mirror image polymerase comprises one or more modifications at one or more amino acid positions disclosed in Table 1 compared to SEQ ID NO: 1. In some embodiments, the mirror image polymerase comprises one or more amino acid substitutions disclosed in Table 1 compared to SEQ ID NO: 1. In some embodiments, the mirror image polymerase comprises all amino acid substitutions disclosed in Table 1. [Table 1-1] [Table 1-2]

[0155] In some embodiments, the mirror polymerase comprises one or more modifications for native chemical ligation (NCL) compared to SEQ ID NO: 1. In some embodiments, the mirror polymerase comprises one or more modifications at one or more amino acid sites selected from E276, K317, N424, and S651 compared to SEQ ID NO: 1. In some embodiments, the mirror polymerase comprises one or more substitutions selected from E276A, K317G, N424A, and S651A compared to SEQ ID NO: 1. In some embodiments, the mirror polymerase comprises substitutions of E276A, K317G, N424A, and S651A compared to SEQ ID NO: 1.

[0156] In some embodiments, the mirror image polymerase comprises one or more isoleucine substitutions compared to SEQ ID NO: 1. In some embodiments, the mirror image polymerase comprises one or more modifications at one or more amino acid sites selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733, and I744 compared to SEQ ID NO: 1. In some embodiments, the mirror image polymerase comprises one or more substitutions of Ile to Ala, Val, Leu, or Tyr at one or more amino acid sites selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733, and I744 compared to SEQ ID NO:1. In some embodiments, the mirror image polymerase comprises one or more substitutions selected from I80V, I127V, I171A, I176V, I191V, I228V, I256V, I264A, I268L, I400V, I597V, I610V, I618A, I630L, I642V, I715Y, I733V, and I744V compared to SEQ ID NO:1.

[0157] In some embodiments, the mirror image polymerase comprises one or more modifications compared to SEQ ID NO: 1 that improve interaction with an L-nucleotide or modifications thereof disclosed herein.

[0158] In some embodiments, the mirror polymerase comprises one or more modifications at one or more amino acid sites selected from M129, I130, G131, D141, E143, L408, Y409, P410, A485, T514, and I521 compared to SEQ ID NO: 1. In some embodiments, the mirror polymerase comprises one or more modifications at one or more amino acid sites selected from D141, E143, Y409, and A485 compared to SEQ ID NO: 1. In some embodiments, the mirror polymerase comprises one or more substitutions selected from D141A, E143A, Y409V, and A485L compared to SEQ ID NO: 1. In some embodiments, the mirror polymerase comprises substitutions of D141A, E143A, Y409V, and A485L compared to SEQ ID NO: 1.

[0159] In some embodiments, the mirror polymerase comprises one or more modifications at one or more amino acid sites selected from D141, E143, L408, Y409, P410, A485, T514, and I521 compared to SEQ ID NO: 1. In some embodiments, the mirror polymerase comprises one or more substitutions selected from D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L compared to SEQ ID NO: 1. In some embodiments, the mirror polymerase comprises substitutions of D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L compared to SEQ ID NO: 1.

[0160] In some embodiments, the mirror image polymerase comprises one or more modifications selected from substitutions of M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S, I521L, or the addition of a D between I130 and G131, compared to SEQ ID NO: 1. In some embodiments, the mirror image polymerase comprises substitutions of M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L, and the addition of a D between I130 and G131, compared to SEQ ID NO: 1.

[0161] In some embodiments, the mirror image polymerase comprises a sequence selected from SEQ ID NOs: 1-7. In some embodiments, the polymerase has a sequence selected from SEQ ID NOs: 1-7.

[0162] The mirror polymerase can be obtained by chemical synthesis. In some embodiments, a polypeptide or small peptide is synthesized by solid-phase peptide synthesis and then ligated by chemical ligation to obtain the mirror polymerase. In some embodiments, the mirror polymerase has the same enzymatic activity as the original polymerase, but acts on D-type nucleotides.

[0163] 5.4.How to use In one aspect, the present disclosure provides a method of using the mirror nucleotide and mirror polymerase disclosed herein.The mirror composition can be used to replicate or sequence L-polynucleotide.In some embodiments, the composition is used in a sequencing method that adopts a sequencing by synthesis method, a Sanger method, or a combination thereof.

[0164] 5.4.1. How to replicate the L-shaped template The present disclosure provides a method for replicating L-polynucleotide. The method may include inducing L-polymerase replication by incubating a mixture containing (i) L-polynucleotide, (ii) L-primer, (iii) L-dNTP, (iv) mirror-type polymerase, and (v) buffer. In some embodiments, the L-polymerase is L-type DNA or RNA. In some embodiments, the L-polymerase contains at least 10, 20, 30, 50, 100, 200, 500 or more nucleotides.

[0165] In some embodiments, the mixture comprises two or more L-dNTPs. In some embodiments, the mixture comprises L-dATP, L-dGTP, L-dCTP, L-dTTP, or modifications thereof. In some embodiments, the mixture comprises L-dATP, L-dGTP, L-dCTP, and L-dTTP.

[0166] In some embodiments, the mixture includes a buffer developed for use with 9°N DNA polymerase. In some embodiments, the buffer includes Tris-HCl at a concentration of 1 mM to 200 mM, 5 mM to 150 mM, 10 mM to 100 mM, 20 mM to 100 mM, or 30 mM to 80 mM. In some embodiments, the buffer includes Tris-HCl at a concentration of about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM. In some embodiments, the buffer includes MgCl2 at a concentration of 1 mM to 100 mM, 5 mM to 100 mM, 10 mM to 100 mM, 15 mM to 50 mM, or 15 mM to 30 mM. In some embodiments, the buffer comprises MgCl2 at a concentration of about 10 mM, 20 mM, 30 mM, 40 mM, or 50 mM. In some embodiments, the buffer comprises MnCl2 at a concentration of 1 mM to 100 mM, 5 mM to 100 mM, 10 mM to 100 mM, 15 mM to 50 mM, or 15 mM to 30 mM. In some embodiments, the buffer comprises MnCl2 at a concentration of about 10 mM, 20 mM, 30 mM, 40 mM, or 50 mM. In some embodiments, the buffer comprises DTT at a concentration of 0.1 mM to 10 mM, 0.5 mM to 5 mM, 0.5 mM to 3 mM, or 0.5 mM to 2 mM. In some embodiments, the buffer comprises DTT at a concentration of about 0.5 mM, 1 mM, 1.5 mM, or 2 mM. In some embodiments, the buffer comprises KCl at a concentration of 10 mM to 100 mM, 20 mM to 80 mM, 25 mM to 75 mM, or 30 mM to 70 mM. In some embodiments, the buffer comprises KCl at a concentration of about 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, or 100 mM. In some embodiments, the buffer has a pH of 7 to 8. In some embodiments, the buffer has a pH of about 7, 7.5, or 8.

[0167] In some embodiments, the buffer comprises Tris-HCl, MgCl2, DTT, and KCl. In some embodiments, the buffer comprises Tris-HCl, MnCl2, DTT, and KCl. In some embodiments, the buffer comprises Tris-HCl, MnCl2, MgCl2, DTT, and KCl. In some embodiments, the buffer comprises 50 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 1 mM DTT, and 50 mM KCl.

[0168] 5.4.2. Sequencing Methods The present disclosure provides a method for sequencing L-polynucleotides. L-polynucleotides include L-form nucleotides. L-polynucleotides can be DNA or RNA.

[0169] In some embodiments, the sequencing method includes the following cycles: (a) obtain a replication product by incubating a mixture containing (i) an L-polynucleotide, (ii) an L-primer, (iii) an L-3'-O-R'-dNTP-R2-label, (iv) a mirror-type polymerase, and (v) a buffer; (b) detect the signal from the L-3'-O-R'-dNTP-R2-label incorporated in the replication product; and (iii) induce the cleavage of the R' and R2 groups of the L-3'-O-R'-dNTP-R2-label incorporated in the replication product. The method includes (i) incorporation of the L-3'-O-R'-dNTP-R2-label, (ii) identification of the incorporated nucleotide by the signal from the incorporated L-3'-O-R'-dNTP-R2-label, and (iii) cleavage of the label and restarting the polymerase reaction to continue sequencing. The L-3'-O-R'-dNTP-R2-label contains a chemical moiety (R') that caps the 3'-OH, and a label tethered to the base via a chemically cleavable linker (R2). The 3' capping moiety (R') and the label on the reaction product are cleaved to restart the polymerase reaction.

[0170] In some embodiments, the method includes multiple cycles. In some embodiments, the cycle is repeated at least 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 times. In some embodiments, the cycle is repeated less than 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, 1000, 2000, 3000, or 5000 times.

[0171] In some embodiments the L-polynucleotide comprises more than 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 nucleotides. In some embodiments the L-polynucleotide comprises less than 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, 1000, 2000, 3000, or 5000 nucleotides.

[0172] In some embodiments, the L-3'-O-R'-dNTP-R2-label comprises an L-3'-O-R'-dATP-R2-label, an L-3'-O-R'-dTTP-R2-label, an L-3'-O-R'-dGTP-R2-label, or an L-3'-O-R'-dCTP-R2-label. In some embodiments, the L-3'-O-R'-dNTP-R2-label comprises two or more L-3'-O-R'-dATP-R2-labels, L-3'-O-R'-dTTP-R2-labels, L-3'-O-R'-dGTP-R2-labels, and L-3'-O-R'-dCTP-R2-labels. In some embodiments, the L-3'-O-R'-dNTP-R2-label includes an L-3'-O-R'-dATP-R2-label, an L-3'-O-R'-dTTP-R2-label, an L-3'-O-R'-dGTP-R2-label, and an L-3'-O-R'-dCTP-R2-label. The labels attached to each type of dNTP can be unique and distinguishable.

[0173] In some embodiments, the L-3'-O-R'-dNTP-R2-label has a structure according to Formula I, described in 5.2.2.1.2. In some embodiments, the L-3'-O-R'-dNTP-R2-label has a structure according to Formula II, described in 5.2.2.1.2.

[0174] In some embodiments, the L-3'-O-R'-dNTP-R2-label comprises a fluorescent label. In this case, the signal is a fluorescent signal. In some embodiments, each type of L-3'-O-R'-dNTP-R2-label (e.g., dATP, dGTP, dTTP, dCTP) is tagged with a unique label. In some embodiments, each type of L-3'-O-R'-dNTP-R2-label (e.g., dATP, dGTP, dTTP, dCTP) provides a unique fluorescent signal. The L-3'-O-R'-dNTP-R2-label may comprise any of the labels disclosed in 5.2.1.3.1. In some embodiments, the L-3'-O-R'-dNTP-R2-label comprises a non-fluorescent label.

[0175] In some embodiments, the sequencing method comprises: (a) obtaining a replication product by incubating a mixture containing (i) L-polynucleotide, (ii) L-primer, (iii) L-dNTP, (iv) L-ddNTP-R2-label, (v) mirror image polymerase disclosed herein, and (vi) buffer; (b) isolating the replication product; and (c) detecting the signal from the L-ddNTP-R2-label incorporated in the replication product. The incubation step can comprise a PCR reaction.

[0176] In a preferred embodiment of the method, a low ratio of L-ddNTP-R2-label is added to the mixture compared to L-dNTP. During incubation (e.g. PCR reaction), the L-ddNTP-R2-label that lacks 3'-OH group can be randomly incorporated into the replication product by mirror image polymerase. The incorporation of L-ddNTP-R2-label terminates the replication process. The reaction can induce the generation of oligonucleotide copies of the replication product that are terminated with random length by L-ddNTP-R2-label.

[0177] In the method, the replication products can be separated by size. In some embodiments, the replication products are separated by size by gel electrophoresis. By detecting the signal from the L-ddNTP-R2-label incorporated in the replication products, the identity of the terminal L-ddNTP-R2-label (e.g., ddATP, ddGTP, ddTTP, or ddCTP) can be determined for the replication products. The data can indicate the type of nucleotide along the length of the L-polynucleotide. This process can allow the determination of the sequence of the L-polynucleotide.

[0178] In some embodiments, L-dNTP comprises L-dATP, L-dTTP, L-dGTP, or L-dCTP. In some embodiments, L-dNTP comprises one or more of L-dATP, L-dTTP, L-dGTP, and L-dCTP. In some embodiments, L-dNTP comprises L-dATP, L-dTTP, L-dGTP, and L-dCTP.

[0179] In some embodiments, L-ddNTP-R2-label comprises L-ddATP-R2-label, L-ddTTP-R2-label, L-ddGTP-R2-label, or L-ddCTP-R2-label.In some embodiments, L-ddNTP-R2-label comprises one or more L-ddATP-R2-label, L-ddTTP-R2-label, L-ddGTP-R2-label, and L-ddCTP-R2-label.In some embodiments, L-ddNTP-R2-label comprises L-ddATP-R2-label, L-ddTTP-R2-label, L-ddGTP-R2-label, and L-ddCTP-R2-label.The label attached to each type of ddNTP can be unique and distinguishable.

[0180] In some embodiments, the L-ddNTP-R2-label has the structure disclosed in 5.2.2.2.1. In some embodiments, the L-ddNTP-R2-label has the formula III defined in 5.2.2.2.1.

[0181] In some embodiments, the L-ddNTP-R2-label comprises a fluorescent label. In this case, the signal is a fluorescent signal. In some embodiments, each type of L-ddNTP-R2-label (e.g., ddATP, ddGTP, ddTTP, ddCTP) is tagged with a unique label. In some embodiments, each type of L-ddNTP-R2-label (e.g., ddATP, ddGTP, ddTTP, ddCTP) provides a unique fluorescent signal. The L-ddNTP-R2-label may comprise any of the labels disclosed in 5.2.1.3. In some embodiments, the L-ddNTP-R2-label comprises a non-fluorescent label.

[0182] In some embodiments, the sequencing method includes the cycle of (a) obtaining a replication product by incubating a mixture containing (i) an L-polynucleotide, (ii) an L-primer, (iii) an L-3'-O-R'-dNTP, (iv) an L-ddNTP-R2-label, (v) a mirror-image polymerase provided herein, and (vi) a buffer; (b) detecting the signal from the L-ddNTP-R2-label incorporated in the replication product; and (c) inducing the cleavage of the R' group of the L-3'-O-R'-dNTP and the R2 group of the L-ddNTP-R2-label incorporated in the replication product. In this method, the polymerase reaction is carried out by a combination of a 3'-capped nucleotide reversible terminator (L-3'-O-R'-dNTP) and a cleavable fluorescent dideoxynucleotide (L-ddNTP-R2-label). In this method, the sequence is determined by the signal of each label on the reaction product terminated by ddNTP (L-ddNTP-R2-label). Upon removal of the 3'-OH capping group (R' group) from the incorporated nucleotide reversible terminator (L-3'-O-R'-dNTP) and the label (L-ddNTP-R2-label) from the ddNTP-terminated DNA product, the polymerase reaction is restarted to continue sequencing.

[0183] In some embodiments, the method includes multiple cycles. In some embodiments, the cycle is repeated at least 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 times. In some embodiments, the cycle is repeated less than 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, 1000, 2000, 3000, or 5000 times.

[0184] In some embodiments the L-polynucleotide comprises more than 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 nucleotides. In some embodiments the L-polynucleotide comprises less than 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, 1000, 2000, 3000, or 5000 nucleotides.

[0185] In some embodiments, the L-3'-O-R'-dNTP comprises L-3'-O-R'-dATP, L-3'-O-R'-dTTP, L-3'-O-R'-dGTP, or L-3'-O-R'-dCTP. In some embodiments, the L-3'-O-R'-dNTP comprises one or more of L-3'-O-R'-dATP, L-3'-O-R'-dTTP, L-3'-O-R'-dGTP, and L-3'-O-R'-dCTP. In some embodiments, the L-3'-O-R'-dNTP comprises L-3'-O-R'-dATP, L-3'-O-R'-dTTP, L-3'-O-R'-dGTP, and L-3'-O-R'-dCTP.

[0186] In some embodiments, the L-3'-O-R'-dNTP has any of the structures provided in 5.2.2.1.1. In some embodiments, the L-3'-O-R'-dNTP has formula I as defined in 5.2.2.1.1.

[0187] In some embodiments, L-ddNTP-R2-label comprises L-ddATP-R2-label, L-ddTTP-R2-label, L-ddGTP-R2-label, or L-ddCTP-R2-label.In some embodiments, L-ddNTP-R2-label comprises one or more L-ddATP-R2-label, L-ddTTP-R2-label, L-ddGTP-R2-label, and L-ddCTP-R2-label.In some embodiments, L-ddNTP-R2-label comprises L-ddATP-R2-label, L-ddTTP-R2-label, L-ddGTP-R2-label, and L-ddCTP-R2-label.The label attached to each type of dNTP can be unique and distinguishable.

[0188] In some embodiments, the L-ddNTP-R2-label has any of the structures provided in 5.2.2.2.1. In some embodiments, the L-ddNTP-R2-label has formula III as defined in 5.2.2.2.1. In some embodiments, each type of L-3'-O-R'-dNTP-R2-label (e.g., dATP, dGTP, dTTP, dCTP) provides a unique fluorescent signal. The L-ddNTP-R2-label can include any of the labels disclosed in 5.2.1.3.1. In some embodiments, the L-ddNTP-R2-label includes a non-fluorescent label.

[0189] In some embodiments, the sequencing method comprises cycles of (a) obtaining reaction products by incubating a mixture that comprises (i) L-polynucleotide, (ii) L-primer, (iii) L-dNTP, (iv) mirror-type polymerase, and (v) buffer; and (b) detecting the release of PPi molecules in the reaction products.In some embodiments, the sequencing method comprises multiple cycles, and in each cycle, the mixture comprises a single type of L-dNTP selected from L-dATP, L-dTTP, L-dGTP, and L-dCTP.In some embodiments, the sequencing method comprises multiple cycles, and each cycle is carried out by stepwise changing the type of L-dNTP to one L-dATP, L-dTTP, L-dGTP, and L-dCTP.

[0190] In some embodiments, detecting the release of the PPi molecule comprises (i) processing the reaction product to convert the PPi molecule ATP, if present, and generate a detectable signal from the ATP, if present; and (ii) detecting the presence or absence of the detectable signal. In some embodiments, the ATP is processed by luciferase to generate a detectable signal. In some embodiments, the detectable signal is light. In some embodiments, the PPi molecule is converted to ATP by ATP-sulfurylase.

[0191] 5.5.Kit The present disclosure provides a kit for replicating or sequencing an L-polymerase, the kit comprising a mirror image polymerase as disclosed herein, and optionally a buffer.

[0192] In some embodiments, the kit further comprises L-dNTP. In some embodiments, the L-NTP comprises L-dATP, L-dGTP, L-dCTP, L-dTTP, or L-UTP. In some embodiments, the L-NTP comprises one or more of L-dATP, L-dGTP, L-dCTP, L-dTTP, and L-UTP. In some embodiments, the L-NTP comprises L-dATP, L-dGTP, L-dCTP, and L-dTTP. In some embodiments, the L-NTP comprises L-dATP, L-dGTP, L-dCTP, and L-UTP. In some embodiments, the L-NTP comprises L-dATP, L-dGTP, L-dCTP, and L-UTP. In some embodiments, the L-NTP comprises L-dATP, L-dGTP, L-dCTP, L-dTTP, and L-UTP.

[0193] In some embodiments, the kit comprises an L-3'-O-R'-dNTP-R2-label, which in some embodiments comprises an L-3'-O-R'-dATP-R2-label, an L-3'-O-R'-dTTP-R2-label, an L-3'-O-R'-dGTP-R2-label, an L-3'-O-R'-dCTP-R2-label, or an L-3'-O-R'-dUTP-R2-label. In some embodiments, the L-3'-O-R'-dNTP-R2-label comprises one or more of an L-3'-O-R'-dATP-R2-label, an L-3'-O-R'-dTTP-R2-label, an L-3'-O-R'-dGTP-R2-label, an L-3'-O-R'-dCTP-R2-label, and an L-3'-O-R'-dUTP-R2-label. In some embodiments, the L-3'-O-R'-dNTP-R2-label comprises one or more of an L-3'-O-R'-dATP-R2-label, an L-3'-O-R'-dTTP-R2-label, an L-3'-O-R'-dGTP-R2-label, and an L-3'-O-R'-dCTP-R2-label. In some embodiments, the L-3'-O-R'-dNTP-R2-label comprises one or more of an L-3'-O-R'-dATP-R2-label, an L-3'-O-R'-dGTP-R2-label, an L-3'-O-R'-dCTP-R2-label, and an L-3'-O-R'-dUTP-R2-label. In some embodiments, each type of L-3'-O-R'-dNTP-R2-label (e.g., dATP, dGTP, dTTP, dCTP) provides a unique fluorescent signal.

[0194] In some embodiments, the L-3'-O-R'-dNTP-R2-label has formula II as defined in 5.2.2.1.2. In some embodiments, the L-3'-O-R'-dNTP-R2-label has any of the structures disclosed in 5.2.2.1.2.

[0195] In some embodiments, the kit comprises L-ddNTP-R2-label. In some embodiments, the L-ddNTP-R2-label comprises L-ddATP-R2-label, L-ddTTP-R2-label, L-ddGTP-R2-label, L-ddCTP-R2-label, or L-ddUTP-R2-label. L-ddNTP-R2-label. In some embodiments, the L-ddNTP-R2-label comprises one or more L-ddATP-R2-label, L-ddTTP-R2-label, L-ddGTP-R2-label, L-ddCTP-R2-label, and L-ddUTP-R2-label. In some embodiments, the L-ddNTP-R2-label comprises L-ddATP-R2-label, L-ddTTP-R2-label, L-ddGTP-R2-label, and L-ddCTP-R2-label. In some embodiments, the L-ddNTP-R2-label comprises an L-ddATP-R2-label, an L-ddGTP-R2-label, an L-ddCTP-R2-label, and an L-ddUTP-R2-label.

[0196] In some embodiments, the L-ddNTP-R2-label has formula III as defined in 5.2.2.2.1. In some embodiments, the L-ddNTP-R2-label has any of the structures disclosed in 5.2.2.2.1.

[0197] In some embodiments, the kit further comprises an L-3'-O-R'-dNTP. The L-3'-O-R'-dNTP may comprise an L-3'-O-R'-dATP, an L-3'-O-R'-dTTP, an L-3'-O-R'-dGTP, or an L-3'-O-R'-dCTP. In some embodiments, the L-3'-O-R'-dNTP comprises one or more of an L-3'-O-R'-dATP, an L-3'-O-R'-dTTP, an L-3'-O-R'-dGTP, and an L-3'-O-R'-dCTP. In some embodiments, the L-3'-O-R'-dNTP comprises an L-3'-O-R'-dATP, an L-3'-O-R'-dTTP, an L-3'-O-R'-dGTP, and an L-3'-O-R'-dCTP.

[0198] In some embodiments, the L-3'-O-R'-dNTP has formula I as defined in 5.2.2.1.1. In some embodiments, the L-3'-O-R'-dNTP has any of the structures disclosed in 5.2.2.1.1.

[0199] In some embodiments, each L-nucleotide-label in the kit has a unique label.In some embodiments, some of the L-nucleotide-labels in the kit have the same label.In some embodiments, the label is a fluorescent label.In some embodiments, each L-nucleotide-label in the kit provides a unique fluorescent signal. EXAMPLES

[0200] 6. Working Example 6.1. Example 1. Synthesis of (L)-3'-O-azidomethyl-dNTP material and method Starting materials beta-L-deoxyadenosine, L-deoxyguanosine, L-deoxycytidine, L-deoxythymidine can be purchased commercially, for example, from Chemgenes. All solvents and reagents were reagent grade, purchased commercially, and used without further purification unless specified. 6.1.1. Synthesis of (L)-3'-O-azidomethyl-dATP 6.1.1.1 Scheme 1. Synthesis of (L)3'-O-N3-dATP [ka]

[0201] A stirred solution of the "starting material" (beta-L-deoxyadenosine) (1.0 equiv., 2.00 g, 7.48 mmol), coevaporated with anhydrous pyridine, is dissolved in 15 ml of anhydrous pyridine and sealed with a septum. After stirring under argon for 5 min, trimethylsilyl chloride (TMS-Cl, 5.0 equiv., 4.06 g, 37.4 mmol) is added via syringe. Benzoyl chloride (1.2 equiv., 1.26 g, 8.98 mmol) is added dropwise via syringe over 20 min and after 30 min, stirring is continued for 2.5 h, during which a clear yellow solution forms. Then 4 ml of H2O is added all at once, and after 5 min, 8 ml of aqueous ammonia (28-30%) is added all at once with stirring for another 15 min. The mixture is evaporated to dryness and the oily residue is coevaporated twice with toluene to give a yellow solid (compound dA-1). To a stirred mixture of compound dA-1 (1.50 g; 3.96 mmol) and imidazole (693 mg; 9.51 mmol) in anhydrous DMF (21.0 mL), tert-butyldimethylsilyl chloride (TBDMSCl) (765 mg; 4.92 mmol) is added. The reaction mixture is stirred at room temperature for 20 h. After evaporation, the residue is purified by flash column chromatography using CH3OH-CH2Cl2 (1:20) as eluent to give compound dA-2 as a white solid. To a stirred solution of compound dA-2 (3.0 g; 6.38 mmol) in DMSO (12 ml), acetic acid (5.5 ml) and acetic anhydride (17.6 ml) are added. The reaction mixture is stirred at room temperature for 48 h. Saturated NaHCO3 solution (100 ml) is added and the aqueous layer is extracted with CHCl2 (3 x 100 ml). The combined organic extracts are washed with saturated NaHCO3 solution (100 ml) and dried over NaSO4. After concentration, the residue is purified by flash column chromatography (hexane / ethyl acetate, 1:1 to 1:4) to give compound dA-3 as a white powder. To a stirred solution of compound dA-3 (400 mg; 0.76 mmol) in dry CHCl2 (7 ml) under nitrogen, cyclohexene (400 μl) and SO2Cl2 (155 μl; 1.91 mmol, redistilled) are added. The reaction mixture is stirred at 0 °C for 2 h. The solvent is removed first under reduced pressure and then under high vacuum pump for 10 min.The residue is dissolved in dry DMF (5 ml) and reacted with NaN3 (400 mg; 6.6 mmol) at room temperature for 3 h. The reaction mixture is dispersed in distilled water (50 ml) and extracted with CHCl2 (3×50 ml). The combined organic layers are dried over NaSO4 and concentrated under reduced pressure. The residue is dissolved in MeOH (5 ml) and stirred with NHF (300 mg; 8.1 mmol) at room temperature for 24 h. The solvent is removed under reduced pressure. The reaction mixture is concentrated under reduced pressure and partitioned between H2O and CHCl2. The organic layer is separated and dried over NaSO4. After concentration, the crude product is purified by flash column chromatography (ethyl acetate / methanol, 100:0 to 98:2) to give compound dA-4 as a white powder. Compound dA-4 (123 mg; 0.3 mmol) and proton sponge (75.8 mg; 0.35 mmol) are dried over PO in a vacuum desiccator overnight and then dissolved in trimethyl phosphate (600 μl). Freshly distilled POCl (40 μl; 0.35 mmol) is then added dropwise at 0 °C and the mixture is stirred at 0 °C for 2 h. A well-vortexed mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 ml; 2.31 mmol) in anhydrous DMF (2.33 ml) is then added in one portion at room temperature and stirred for 30 min. Triethylammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 ml) is then added and the mixture is stirred at room temperature for 1 h. Concentrated NHOH (15 ml) is then added and stirred at room temperature overnight. The resulting mixture is concentrated under vacuum and the residue is diluted with 5 ml of water. The crude mixture is then purified by anion exchange chromatography on DEAE-Sephadex A-25 using a gradient of TEAB (pH 8.0; 0.1-1.0 M) at 4° C. The crude product is further purified by reverse-phase HPLC to give (L) 3′-O-azidomethyl-dATP (compound dA-5). 6.1.2.(L) Synthesis of 3'-O-azidomethyl-dGTP 6.1.2.1 Scheme 2. Synthesis of (L)3'-O-N3-dGTP [ka]

[0202] A stirred solution of the "starting material" (beta-L-deoxyadenosine) (1.0 equiv., 2.00 g, 7.13 mmol) is coevaporated with anhydrous pyridine (3 x 4 mL) and then dissolved in anhydrous pyridine (2 mL). The resulting solution is protected from moisture (drying tube), purged with argon, and placed on ice. To the ice-cold solution, TMS-Cl (4.51 mL, 58.4 mmol, 8.2 equiv.) is added dropwise via syringe. The ice bath is then removed and the mixture is stirred for 2 h. The solution is cooled on ice and isobutyric anhydride (0.29 mL, 15.69 mmol, 2.2 equiv.) is added dropwise via syringe and the ice bath is removed. After stirring at room temperature for another 2 h, the reaction is again placed on ice and ice-cold water (20 mL) is slowly added followed after 15 min by concentrated ammonia solution (1.5 mL) to obtain a final concentration of ammonia of 2.5 M. The mixture is kept on ice for 30 min and then evaporated to dryness. The residue is coevaporated with toluene (3 × 5 mL) to remove traces of water, resuspended in MeOH, and filtered to remove precipitate. The filtrate is then concentrated, dissolved in a small amount of MeOH, absorbed onto silica gel, and purified by column chromatography (DCM / MeOH 95:5 to 91:9 (v / v)) to give compound dG-1 as a yellow solid. Compound dG-1 (495 mg, 1.07 mmol) is coevaporated with dry pyridine three times, dried under high vacuum, and washed with 2 cm water in an ice bath. 3The mixture is dissolved in dry N,N-dimethylformamide. Di-tert-butylsilyl bis(trifluoromethanesulfonate) (590 mg, 1.34 mmol) is added dropwise over 15 min and the reaction mixture is stirred at 0° C. for 30 min. Imidazole (419 mg, 6.15 mmol) is added and the mixture is stirred at 0° C. for 15 min and at room temperature for 15 min. Then 241 mg of tert-butyldimethlsiliyl chloride (1.59 mmol) is added and the solution is stirred at 60° C. for another 2 h. The mixture is diluted with dichloromethane, washed with brine, dried over sodium sulfate and evaporated. The crude product is purified by column chromatography on silica gel (methanol:dichloromethane 0:100 to 2:98) as a white foam (compound dG-2). To a stirred solution of compound dG-2 (3.0 g; 6.08 mmol) in DMSO (12 ml), acetic acid (5.5 ml) and acetic anhydride (17.6 ml) are added. The reaction mixture is stirred at room temperature for 48 h. Saturated NaHCO3 solution (100 ml) is added and the aqueous layer is extracted with CHCl2 (3 x 100 ml). The combined organic extracts are washed with saturated NaHCO3 solution (100 ml) and dried over NaSO4. After concentration, the residue is purified by flash column chromatography (hexane / ethyl acetate, 1:1 to 1:4) to give compound dG-3 as a white powder. To a stirred solution of compound dG-3 (1.0 g; 2.0 mmol) in dry pyridine (22 ml), diphenylcarbamoyl chloride (677 mg; 2.92 mmol) and DIEA (N,N-diisopropylethylamine) (1.02 ml; 5.9 mmol) are added. The reaction mixture is stirred at room temperature under nitrogen for 3 h. The solvent is removed under high vacuum. The crude product is purified by flash column chromatography (ethyl acetate / hexane, 1:1 to 7:3) to give compound dG-4 as a yellowish powder. To a stirred solution of compound dG-4 (400 mg; 0.71 mmol) in dry CHCl (7 ml) under nitrogen, cyclohexene (400 μl) and SOCl (155 μl; 1.91 mmol, redistilled) are added. The reaction mixture is stirred at 0 °C for 2 h.The solvent is removed first under reduced pressure and then under high vacuum pump for 10 min. The residue is dissolved in dry DMF (5 ml) and reacted with NaN3 (400 mg; 6.6 mmol) at room temperature for 3 h. The reaction mixture is dispersed in distilled water (50 ml) and extracted with CHCl2 (3 x 50 ml). The combined organic layers are dried over NaSO4 and concentrated under reduced pressure. The residue is dissolved in MeOH (5 ml) and stirred with NHF (300 mg; 8.1 mmol) at room temperature for 24 h. The solvent is removed under reduced pressure. The reaction mixture is concentrated under reduced pressure and partitioned between H2O and CHCl2. The organic layer is separated and dried over NaSO4. After concentration, the crude product is purified by flash column chromatography (ethyl acetate / methanol, 100:0 to 98:2) to give compound dG-5 as a white powder. Compound dG-5 (123 mg; 0.28 mmol) and proton sponge (75.8 mg; 0.35 mmol) are dried over PO in a vacuum desiccator overnight and then dissolved in trimethyl phosphate (600 μl). Freshly distilled POCl (40 μl; 0.35 mmol) is then added dropwise at 0 °C and the mixture is stirred at 0 °C for 2 h. A well-vortexed mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 ml; 2.31 mmol) in anhydrous DMF (2.33 ml) is then added in one portion at room temperature and stirred for 30 min. Triethylammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 ml) is then added and the mixture is stirred at room temperature for 1 h. Concentrated NHOH (15 ml) is then added and stirred at room temperature overnight. The resulting mixture is concentrated under vacuum and the residue is diluted with 5 ml of water. The crude mixture is then purified by anion exchange chromatography on DEAE-Sephadex A-25 at 4° C. using a gradient of TEAB (pH 8.0; 0.1-1.0 M). The crude product is further purified by reverse-phase HPLC to give (L) 3′-O-azidomethyl-dGTP (compound dG-6). 6.1.3.(L) Synthesis of 3'-O-azidomethyl-dCTP 6.1.3.1 Scheme 3. Synthesis of (L)3'-O-N3-dCTP [ka]

[0203] The procedure for the preparation of (L)3'-O-azidomethyl-dCTP (compound dC-5) is similar to the synthetic protocol (5 steps) outlined above for (L)3'-O-azidomethyl-dATP. The starting material for the synthesis of (L)3'-O-azidomethyl-dCTP is beta-L-deoxycytidine (Chemgenes). 6.1.4.(L) Predictive Synthesis of 3'-O-Azidomethyl-dTTP 6.1.4.1 Scheme 4. Synthesis of (L)3'-O-N3-dTTP [ka]

[0204] To a stirred mixture of "starting material" (beta-L-deoxythymidine) (1.3 g; 3.87 mmol) and imidazole (693 mg; 9.51 mmol) in anhydrous DMF (21.0 mL), tert-butyldimethylsilyl chloride (TBDMSCl) (765 mg; 4.92 mmol) is added. The reaction mixture is stirred at room temperature for 20 h. After evaporation, the residue is purified by flash column chromatography using CH3OH-CH2Cl2 (1:20) as eluent to give compound dT-1 as a white solid. To a stirred solution of compound dT-1 (3.0 g; 6.38 mmol) in DMSO (12 ml), acetic acid (5.5 ml) and acetic anhydride (17.6 ml) are added. The reaction mixture is stirred at room temperature for 48 h. Saturated NaHCO3 solution (100 ml) is added and the aqueous layer is extracted with CHCl (3×100 ml). The combined organic extracts are washed with saturated NaHCO3 solution (100 ml) and dried over NaSO. After concentration, the residue is purified by flash column chromatography (hexane / ethyl acetate, 1:1 to 1:4) to obtain compound dT-2 as a white powder. To a stirred solution of compound dT-2 (400 mg; 0.76 mmol) in dry CHCl (7 ml) under nitrogen, cyclohexene (400 μl) and SOCl (155 μl; 1.91 mmol, redistilled) are added. The reaction mixture is stirred at 0° C. for 2 h. The solvent is removed first under reduced pressure and then under high vacuum pump for 10 min. The residue is dissolved in dry DMF (5 ml) and reacted with NaN (400 mg; 6.6 mmol) at room temperature for 3 h. The reaction mixture is dispersed in distilled water (50 ml) and extracted with CHCl (3×50 ml). The combined organic layers are dried over NaSO and concentrated under reduced pressure. The residue is dissolved in MeOH (5 ml) and stirred with NHF (300 mg; 8.1 mmol) at room temperature for 24 h. The solvent is removed under reduced pressure. The reaction mixture is concentrated under reduced pressure and partitioned between H0 and CHCl. ​​The organic layer is separated and dried over NaSO. After concentration, the crude product is purified by flash column chromatography (ethyl acetate / methanol, 100:0 to 98:2) to give compound dT-3 as a white powder.Compound dT-3 (123 mg; 0.3 mmol) and proton sponge (75.8 mg; 0.35 mmol) are dried over PO in a vacuum desiccator overnight and then dissolved in trimethyl phosphate (600 μl). Freshly distilled POCl (40 μl; 0.35 mmol) is then added dropwise at 0 °C and the mixture is stirred at 0 °C for 2 h. A well-vortexed mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 ml; 2.31 mmol) in anhydrous DMF (2.33 ml) is then added in one portion at room temperature and stirred for 30 min. Triethylammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 ml) is then added and the mixture is stirred at room temperature for 1 h. Concentrated NHOH (15 ml) is then added and stirred at room temperature overnight. The resulting mixture is concentrated under vacuum and the residue is diluted with 5 ml of water. The crude mixture is then purified by anion exchange chromatography on DEAE-Sephadex A-25 using a gradient of TEAB (pH 8.0; 0.1-1.0 M) at 4° C. The crude product is further purified by reverse-phase HPLC to give (L) 3′-O-azidomethyl-dTTP (compound dT-4).

[0205] 6.2. Example 2. Synthesis of (L)3'-O-R'-dNTP Those skilled in the art who have reviewed the exemplary methods provided herein will be able to easily apply these methods to generate compounds with various R' groups that protect 3'O. Specifically, allyl and nitrobenzyl analogs of the described (L)-3'-O-azidomethyl-dNTPs are contemplated, which utilize allyl bromide and 2-nitrobenzyl bromide, respectively, instead of the acetic acid / acetic anhydride step in the previous reaction schemes 1-4. Specifically, reference is made to the methods provided in Ju J. et al. 2006, PNAS, vol.103, No.52 and Wu J. et al. 2007, PNAS, vol.104, No.42, respectively, the entire contents of each of which, including supporting information, are incorporated herein by reference. 6.3. Example 3. (L) Synthesis of 3'-O-azidomethyl-dNTP-labeled 6.3.1. Synthesis of (L)-3'-O-azidomethyl-dATP-ROX 6.3.1.1 Scheme 5. Synthesis of NH2-(L)3'-O-N3-dATP [ka] 6.3.1.2 Scheme 6. (L) Synthesis of 3'-O-N3-dATP-ROX [ka]

[0206] Azido-ROX compound (ROX-N3-linker). (2-{2-[3-(2-amino-ethylcarbamoyl)-phenoxy]-1-azido-ethoxy}-ethoxy)-acetic acid linker-6 (7.0 mg, 0.019 mmol), prepared according to the literature (Milton J, Ruediger S, Liu X (2006), US Patent Publication No. 20060160081), is dissolved in DMF (300 μl) and 1M aqueous NaHCO3 (100 μl). A solution of ROX NHS (N-hydroxysuccinimide) ester (Invitrogen) (0.013 mmol) in DMF (400 μl) is slowly added to the previous reaction mixture, which is then stirred at room temperature for 5 hours in the absence of light. The crude product is purified on a preparative silica gel TLC plate (CHCl3 / CH3OH, 1:4).

[0207] Compound L-3'-O-azidomethyl-dATP-ROX (L-3'-O'-N3-dATP-ROX). To a stirred solution of ROX-N3-linker in dry DMF (2 ml), DSC (N,N'-disuccinimidyl carbonate) (3.4 mg, 13.2 μmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 μmol) are added. The reaction mixture is stirred for 2 h at room temperature. TLC shows that the ROX-N3-linker is completely converted to the compound ROX-N3-linker NHS ester, which is used directly to couple with L-amino-dATP (13 μmol) in NaHCO3 / Na2CO3 buffer (pH 8.7, 0.1 M) (300 μl). The reaction mixture is stirred for 3 h at room temperature with the exclusion of light. The reaction mixture is purified by preparative silica gel TLC plate (CHOH / CHCl, 1:1). The crude product is further purified by reverse phase HPLC to give L-3'-O-azidomethyl-dATP-ROX (L-3'-O-N-dATP-ROX). 6.3.2.(L) Synthesis of 3'-O-azidomethyl-dGTP-Cy5 6.3.2.1 Scheme 7. Synthesis of NH2-(L)3'-O-N3-dGTP [ka] 6.3.2.2 Scheme 8. (L)3'-O-N3-dGTP-Cy5 [ka]

[0208] Azide-Cy5 compound (Cy5-N3-Linker). (2-{2-[3-(2-amino-ethylcarbamoyl)-phenoxy]-1-azido-ethoxy}-ethoxy)-acetic acid linker-6 (7.0 mg, 0.019 mmol), prepared according to the literature (Milton J, Ruediger S, Liu X (2006), US Patent Publication No. 20060160081), is dissolved in DMF (300 μl) and 1M NaHCO3 aqueous solution (100 μl). A solution of Cy5 NHS (N-hydroxysuccinimide) ester (Invitrogen) (0.013 mmol) in DMF (400 μl) is slowly added to the previous reaction mixture, which is then stirred at room temperature for 5 hours in the absence of light. The crude product is purified on a preparative silica gel TLC plate (CHCl3 / CH3OH, 1:4).

[0209] Compound L-3'-O-azidomethyl-dGTP-Cy5 (L-3'-O-N3-dGTP-Cy5). To a stirred solution of Cy5-N3-linker in dry DMF (2 ml), DSC (N,N'-disuccinimidyl carbonate) (3.4 mg, 13.2 μmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 μmol) are added. The reaction mixture is stirred for 2 h at room temperature. TLC shows that the Cy5-N3-linker is completely converted to the compound Cy5-N3-linker NHS ester, which is used directly to couple with L-amino-dGTP (13 μmol) in NaHCO3 / Na2CO3 buffer (pH 8.7, 0.1 M) (300 μl). The reaction mixture is stirred for 3 h at room temperature with the exclusion of light. The reaction mixture is purified by preparative silica gel TLC plate (CHOH / CHCl, 1:1). The crude product is further purified by reverse phase HPLC to give L-3'-O-azidomethyl-dGTP-Cy5 (L-3'-O-N3-dGTP-Cy5). 6.3.3. (L) Synthesis of 3'-O-azidomethyl-dCTP-Bodipy-FL-510 6.3.3.1 Scheme 9. Synthesis of NH2-(L)3'-O-N3-dCTP [ka] 6.3.3.2 Scheme 10. Synthesis of (L)3'-O-N3-dCTP-Bodipy-FL-510 [ka]

[0210] Azido-Bodipy-FL-510 (compound BODIPY-FL-510-N3-Linker). (2-{2-[3-(2-amino-ethylcarbamoyl)-phenoxy]-1-azido-ethoxy}-ethoxy)-acetic acid linker-6 (7.0 mg, 0.019 mmol), prepared according to the literature (Milton J, Ruediger S, Liu X (2006), US Patent Publication No. 20060160081), is dissolved in DMF (300 μl) and 1M aqueous NaHCO3 (100 μl). A solution of Bodipy-FL-510 NHS (N-hydroxysuccinimide) ester (Invitrogen) (5.0 mg, 0.013 mmol) in DMF (400 μl) is slowly added to the previous reaction mixture, which is then stirred at room temperature for 5 hours in the absence of light. The crude product is purified on a preparative silica gel TLC plate (CHCl3 / CH3OH, 1:4) to give the BODIPY-FL-510-N3-linker.

[0211] L-3'-O-azidomethyl-dCTP-Bodipy-FL-510 (compound L-3'-O-N3-dCTP-Bodipy-FL-510). To a stirred solution of BODIPY-FL-510-N3-linker in dry DMF (2 ml) is added DSC (N,N'-disuccinimidyl carbonate) (3.4 mg, 13.2 μmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 μmol). The reaction mixture is stirred at room temperature for 2 h. TLC shows that BODIPY-FL-510-N3-linker is completely converted to the compound BODIPY-FL-510-N3-linker NHS ester, which is used directly to couple with L-amino-dCTP (13 μmol) in NaHCO3 / Na2CO3 buffer (pH 8.7, 0.1 M) (300 μl). The reaction mixture is stirred at room temperature for 3 hours with the exclusion of light. The reaction mixture is purified by preparative silica gel TLC plate (CH3OH / CH2Cl2, 1:1). The crude product is further purified by reverse phase HPLC to obtain L-3'-O-azidomethyl-dCTP-Bodipy-FL-510. 6.3.4.(L) Synthesis of 3'-O-azidomethyl-dUTP-R6G 6.3.4.1 Scheme 11. Synthesis of NH2-(L)3'-O-N3-dUTP [ka] 6.3.4.2 Scheme 12. Synthesis of (L)3'-O-N3-dUTP-R6G [ka]

[0212] Azide-R6G (compound R6G-N3-linker). (2-{2-[3-(2-amino-ethylcarbamoyl)-phenoxy]-1-azido-ethoxy}-ethoxy)-acetic acid (7.0 mg, 0.019 mmol), prepared according to the literature (Milton J, Ruediger S, Liu X (2006), US Patent Publication No. 20060160081), is dissolved in DMF (300 μl) and 1M aqueous NaHCO3 (100 μl). A solution of R6G NHS (N-hydroxysuccinimide) ester (Invitrogen) (0.013 mmol) in DMF (400 μl) is slowly added to the previous reaction mixture, which is then stirred for 5 hours at room temperature under the exclusion of light. The crude product is purified on a preparative silica gel TLC plate (CHCl3 / CH3OH, 1:4).

[0213] L-3'-O-azidomethyl-dUTP-R6G. To a stirred solution of R6G-N3-linker in dry DMF (2 ml), DSC (N,N'-disuccinimidyl carbonate) (3.4 mg, 13.2 μmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 μmol) are added. The reaction mixture is stirred at room temperature for 2 hours. TLC shows that the R6G-N3-linker is completely converted to the compound R6G-N3-linker NHS ester, which is used directly to couple with L-amino-dUTP (13 μmol) in NaHCO3 / Na2CO3 buffer (pH 8.7, 0.1 M) (300 μl). The reaction mixture is stirred at room temperature for 3 hours with the exclusion of light. The reaction mixture is purified by preparative silica gel TLC plate (CH3OH / CH2Cl2, 1:1). The crude product is further purified by reverse phase HPLC to give L-3'-O-azidomethyl-dUTP-R6G (L-3'-O-N3-dUTP-R6G).

[0214] 6.4. Example 4. Synthesis of (L) 3'-O-allyl-dNTP-allyl-label Those skilled in the art who have reviewed the exemplary methods provided herein can easily apply the methods provided to generate compounds with various R' groups that protect 3'O, and alternative cleavable linkers that incorporate, for example, the allyl-fluorophore linker reported in Ju J.et al. 2006, PNAS, vol.103, No.52, according to the following scheme. Specifically, the allyl analog of the described (L)-3'-O-azidomethyl-dNTP-NH2 compound is contemplated, which utilizes allyl bromide instead of the acetic acid / acetic anhydride step in the previous reaction schemes 5, 7, 9, and 11. Specifically, reference is made to the method provided in Ju J.et al. 2006, PNAS, vol.103, No.52, the entire contents of which, including supporting information, are incorporated herein by reference. [ka] 1. Synthesis of the Allyl-Linker [ka]

[0215] 6.5. Example 5. Synthesis of Mirror Image Polymerase The amino acid sequence of 9°N polymerase is divided into multiple segments. Each polypeptide segment is synthesized using solid-phase peptide synthesis (Fmoc-SPPS), a strategy based on the use of 9-fluorenylmethoxycarbonyl (Fmoc) as a protecting group. Once the polypeptide segments are synthesized, they are separated and purified using semi-preparative reversed-phase high performance liquid chromatography (RP-HPLC). The separated polypeptide segments are ligated from the C-terminus to the N-terminus using native chemical ligation methods. After the chemical ligation reaction is completed, the target product is isolated using semi-preparative RP-HPLC.

[0216] The synthesized mirror-image polymerase is processed for folding and renaturation, and circular dichroism and mass spectrometry are used to confirm that the mirror-image polymerase is correctly folded.

[0217] 6.6. Example 6. Mirror-Image Polymerase Activity The mirror polymerase is mixed with (i) polymerase reaction buffer; (ii) L-primer; (iii) L-polynucleotide template, and (iv) four 0.4 mM (L) dNTPs. The reaction mixture is placed at 37° C. for 4 hours, and the reaction is terminated by adding 1 μl of 0.5 M EDTA. The activity of the mirror polymerase is measured based on the yield of polynucleotide fragments complementary to the template, or by using a double-stranded DNA intercalating dye (e.g., EvaGreen dye) and measuring the increase in fluorescence.

[0218] 6.7. Example 7. Mirror-Image Polymerase Chain Reaction The mirror image polymerase is mixed with (i) polymerase reaction buffer; (ii) 2.5 μM L-primer; (iii) 2.5 μM L-polynucleotide template, and (iv) four 0.4 mM (L) dNTPs. The first cycle consisted of 5 min at 95° C., 5 min at 50° C. (during which the polymerase and BSA are added), and 5 min at 70° C. The segments of each subsequent PCR cycle are as follows: 1 min at 93° C., 1 min at 50° C., and 5 min at 70° C. After 0, 13, 23, and 40 cycles, 20 μl volumes of 100 μl are removed and subjected to agarose gel electrophoresis with ethidium bromide present to quantify the amplification of the template sequence. The reaction produces polynucleotide fragments complementary to the template.

[0219] 6.8. Example 8. General Procedure for Specific Single-Base Extension, Deprotection, and Reinitiation of L-Primer Extension Using (L)-3'-OR-dNTPs, (L)-3'-OR-dNTP-R-Labeled, (L)-ddNTP-R2-Labeled, and -D-Polymerase to Demonstrate Sequencing by Synthesis (SBS) To obtain de novo DNA sequencing data for the L-primer / L-DNA template immobilized on the solid surface, first, in each polymerase extension reaction, a verification of accurate and specific single-base extension is performed using a combination mixture of solution A consisting of 3'-O-N3-dCTP (3 μM), 3'-O-N3-dTTP (3 μM), 3'-O-N3-dATP (3 μM), and 3'-O-N3-dGTP (0.5 μM), and solution B consisting of ddCTP-N3-Bodipy-FL-510 (50 nM), ddUTP-N3-R6G (100 nM), ddATP-N3-ROX (200 nM), and ddGTP-N3-Cy5 (100 nM). For example, 60 pmol of self-priming DNA template, 1x Thermopol II reaction buffer, 40 nmol of MnCl2, and 1 unit of D-polymerase are added together with modified L-nucleotide reversible terminator in a total reaction volume of 20 μl. The reaction consisted of incubation at 94°C for 5 minutes, 4°C for 5 minutes, and 65°C for 20 minutes. The extension products are then analyzed by fluorescence-based gel electrophoresis (in addition, MALDI-TOF MS can be used as an alternative option) to confirm the specific incorporation of the correct nucleotide. For cleavage of DNA extension products bearing 3'-O-N3-dNTP and ddNTP-N3-fluorophores, the DNA products are resuspended in 50 μl of 100 mM TCEP solution (pH 9.0) at 65° C. for 15 min and then analyzed by either gel electrophoresis or MALDI-TOF MS to confirm cleavage of the R label / protecting group, allowing reinitiation of extension of the next base. The above describes a complete single cycle of SBS (extension, label detection, and cleavage).

[0220] In general, separate solutions of "solution A" consisting of four L-3'-O-R'-dNTPs (each with dATP, dTTP, dGTP, or dCTP) and "solution B" consisting of four L-ddNTP-R2-labels (each with ddATP, ddTTP, ddGTP, or ddCTP) are used for polymerase extension reaction. Solution A and solution B are mixed with mirror polymerase, L-primer, buffer, and L-polynucleotide template in a specific ratio (i.e., 7:3 v / v, 9:1 v / v), and the mixture is incubated for multiple cycles of sequence by synthesis (SBS). During the SBS cycles, mirror polymerase synthesizes complementary sequences to L-polynucleotides using a combination of 3'-capped nucleotide reversible terminators (L-3'-O-R'-dNTPs) and cleavable fluorescent dideoxynucleotides (L-ddNTP-R2-labels). The replication product terminated by ddNTP (L-ddNTP-R2-label) is detected using the signal from the label specific to ddATP, ddTTP, ddGTP, or ddCTP. After the signal is detected, the 3'-OH capping group (R' group) is cleaved from the incorporated nucleotide reversible terminator (L-3'-O-R'-dNTP) and the label (L-ddNTP-R2-label) is cleaved from the DNA product terminated by ddNTP, and the polymerase reaction is restarted. Since each label conjugated to dATP, dTTP, dGTP, or dCTP is unique, the fluorescent signal from the label indicates the nucleotide corresponding to the termination site. Based on the signal, the sequence of the L-polynucleotide template is determined.

[0221] Also, the L-polynucleotide template is sequenced using four kinds of L-3'-O-R'-dNTP-R2-labels (each having dATP, dTTP, dGTP, or dCTP). The L-polynucleotide template is mixed with four kinds of L-3'-O-R'-dNTP-R2-labels (each having dATP, dTTP, dGTP, or dCTP), L-primer, D-polymerase, and buffer under the same reaction conditions as above. The mixture is incubated for multiple cycles of sequencing by synthesis (SBS). During the SBS step, the L-3'-O-R'-dNTP-R2-label is incorporated into the synthesis product. The L-3'-O-R'-dNTP-R2-label comprises a chemical moiety (R') that caps the 3'-OH, and a label that is connected to the base via a chemically cleavable linker (R2). After incorporation, the fluorescent signal from the label is detected, and then the 3' capping portion (R') and the label on the reaction product are cleaved to restart the polymerase reaction. Since each label conjugated to dATP, dTTP, dGTP, or dCTP is unique, the fluorescent signal from the label indicates the nucleotide corresponding to the termination site. Based on the signal, the sequence of the L-polynucleotide template is determined.

[0222] 6.9. Example 9. Sequencing of L-polynucleotides by methods similar to Sanger sequencing Four kinds of L-ddNTP-R2-labels (each with ddATP, ddTTP, ddGTP, or ddCTP) are used to sequence the L-polynucleotide template (similar to Sanger sequencing). The four kinds of L-ddNTP-R2-labels are mixed with the L-polynucleotide template, the L-primer, the four kinds of L-dNTPs (L-dATP, L-dGTP, L-dCTP, and L-dTTP), D-polymerase, and buffer under the same reaction conditions as above. In the mixture, the L-ddNTP-R2-labels are added to the mixture in a much smaller amount compared to the L-dNTPs. The mixture is used to carry out PCR (cycle sequencing-generating DNA fragment ladder). The replication products are separated by size using gel electrophoresis to detect the signal from the L-ddNTP-R2-labels incorporated in the replication products. Since each label conjugated to dATP, dTTP, dGTP, or dCTP is unique, the fluorescent signal from the label indicates the nucleotide corresponding to the termination site. Based on the fluorescent signal and fragment mobility (based on size), the sequence of the L-polynucleotide template is determined.

[0223] 6.10. Example 10. Complete synthesis of (L)-3'-O-azidomethyl-dNTP Four target molecules and their synthetic routes were designed as shown in Schemes 13-16. L-3'-O-azidomethyl-dTTP (dT-4) and L-3'-O-azidomethyl-dCTP (dC-5) were prepared to 1 H, 31 The compounds were characterized by P NMR, and HRMS. For the synthesis of L-3'-O-azidomethyl-dATP (dA-5) and L-3'-O-azidomethyl-dGTP (dG-6), we synthesized intermediates dA-3 and dG-5, respectively.

[0224] material and method All solvents and reagents were reagent grade, purchased commercially and used without further purification unless specified. All chemicals were purchased from Sigma-Aldrich, Fisher Scientific, TCI, etc. 1H NMR spectra were recorded on a Bruker Ascend™ (400 MHz) spectrometer at Chapman University and reported in parts per million (ppm) from CDCl3 (7.26 ppm) or D2O. Data were reported as follows: (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets, J = coupling constant (Hz), integral). Proton decoupled 31 P NMR spectra were recorded on a Bruker Ascend™ (121.4 MHz) spectrometer at Chapman University. High-resolution mass spectra (HRMS) were obtained from the Analytical Chemistry Instrumentation Facility at the School of Pharmacy, Chapman University, and the University of California at Riverside. Starting materials beta-L-deoxythymidine, beta-L-deoxycytidine, beta-L-deoxyadenosine, and beta-L-deoxyguanosine were purchased from Chemgenes. Analytical (Polaris 180A C18-A, 4.6×250 mm, 5 μm) and semi-preparative (Polaris 180A C18-A, 4.6×250 mm, 5 μm) HPLC columns were purchased from Agilent. 3'-O-modified nucleotides were purified by reversed-phase HPLC on a 4.6 x 250 mm C18 column (Polaris), mobile phase: A, 25 mM TEAB buffer in water; B, 25 mM TEAB buffer in acetonitrile. Elution was performed under isocratic conditions as described in each procedure. 6.10.1.(L) Synthesis of 3'-O-azidomethyl-dTTP 6.10.1.1 Scheme 13. (L) Synthesis of 3'-O-N3-dTTP (dT-4) [ka]

[0225] Testing Procedure: Synthesis of dT-1: To a solution of β-L-deoxythymidine (1.5 g, 6.18 mmol) in anhydrous N,N-dimethylformamide (DMF) (37.5 mL) was added imidazole (633 mg, 9.30 mmol) and tert-butyldimethylsilyl chloride (1.02 g, 6.81 mmol) under nitrogen at 0°C. After stirring at room temperature for 3 h, ice-cold water was added to the solution and extracted with EtOAc (2 x 50 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated. The resulting residue was dissolved in CH3OH and silica gel was added. The solution was evaporated under reduced pressure until dry. The resulting residue was purified by column chromatography (2:98 to 5:95, CH3OH-CH2Cl2) to give dT-1 (1.8 g, 82%) as a white solid. R f 0.35 (1:19 CH3OH-CH2Cl2). The product was confirmed by TLC.

[0226] dT-2 synthesis: To a stirred solution of dT-1 (1.9 g; 5.33 mmol) in DMSO (18 ml) was added acetic acid (9 ml) and acetic anhydride (27 ml) at room temperature. The reaction mixture was stirred at room temperature for 48 h. Saturated NaHCO3 solution was added at 0° C., stirred for 30 min, and the aqueous layer was extracted with CH2Cl2 (2×100 ml). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by flash column chromatography (1:1, hexane-EtOAc) to give dT-2 as a yellowish syrup (2.0 g, 90%). R f 0.55 (1:1 hexane-EtOAc); 1 H NMR(400MHz,CDCL3):δ9.36(s,1H,NH),7.48(d,1H, 4 J=1.1Hz,H-6),6.31(dd,1H,J 1’,2’a =5.6Hz,J 1’,2’b =8.6Hz,H-1'),4.68(d,1H,J gem =11.8Hz,CH2S),4.61(d,1H,J gem =11.8Hz,CH2S),4.47(app dt,1H,J 3’,2’b =5.9Hz,J3’,2’a = 1.9 Hz, (H-3’), 4.12 - 4.08 (m, 1H, H-4’), 3.89 (dd, 1H, J 5’a,4’ = 2.6 Hz, J gem = 11.3 Hz, (H-5’a), 3.80 (dd, 1H, J 5’a,4’ = 2.9 Hz, J gem = 11.3 Hz, (H-5’b), 2.41 (ddd, 1H, J 2’a,3’ = 1.9 Hz, J 2’a,1’ = 5.6 Hz, J gem = 13.6 Hz, (H-2’a), 2.16 (s, 3H, SCH3), 1.99 (ddd, 1H, J 2’b,3’ = 5.9 Hz, J 2’b,1’ = 8.6 Hz, J gem = 13.6 Hz, (H-2’b), 1.92 (d, 3H, 4 J = 1.1 Hz, CH3), 0.93 (s, 9H, (CH3)3CSi), 0.12 (s, 6H, (CH3)2Si)); HRMS(ESI) m / z [M + H] + C 18 H 33 Calculated value for C

Chemical Structure

[0227] Synthesis of dT-3: To a stirred solution of dT-2 (2.0 g, 4.80 mmol) in dry CHCl (45 mL) was added cyclohexene (2.1 mL) and SOCl (1.0 M in DCM) (6.48 mL, 6.48 mmol). The reaction mixture was stirred at 0 °C for 3 h. The volatiles were removed under reduced pressure. The residue was dissolved in dry DMF (30 mL) and reacted with NaN (1.88 g, 28.8 mmol) at room temperature for 2 h. The reaction mixture was dispersed in cold distilled water (100 mL) and extracted with EtOAc (2 × 200 mL). The combined organic extracts were dried over NaSO and concentrated under reduced pressure. The resulting residue was dissolved in CHCN (10 mL) and reacted with 2 M HCl (2-3 mL) at 0 °C for 5 h. Saturated Na2CO3 solution was added, extracted with CH2Cl2 (2 x 50 mL), dried over Na2SO4, and concentrated. The organic layer was washed with water, and the organic layer was dried over Na2SO4 and concentrated. The resulting residue was purified by flash column chromatography (hexane / ethyl acetate, 3:7 to 1:4) to give 3 as a white powder. R f 0.20 (1:4 hexane-EtOAc); 1 H NMR(400MHz,CDCL3):δ8.63(s,1H,NH),7.38(d,1H, 4 J=1.1Hz,H-6),6.13(app t,1H,J 1’,2’a =J 1’,2’b =7.0Hz,H-1'),4.77(d,1H,J gem =9.0Hz,CH2N3),4.70(d,1H,J gem =9.0Hz,CH2N3),4.50(app dt,1H,J 3’,2’b =6.0Hz,J 3’,2’a =3.5Hz,H-3'),4.16-4.12(m,1H,H-4'),3.98(dd,1H,J 5’a,4’ =2.7Hz,J gem =12.0Hz,H-5'a),3.84(dd,1H,J 5’a,4’ =2.8Hz,J gem =12.0Hz,H-5'b),2.51-2.38(m,3H,H-2'a,H-2'b,5'-OH),1.94(d,3H, 4J=1.1Hz,CH3);HRMS(ESI)m / z [M+H] + C 11 H 16 Calculated value of N5O5S: 298.1151; Measured value: 298.1144 [ka]

[0228] dT-4 synthesis: dT-3 (120 mg, 0.403 mmol) was dried over PO in a vacuum desiccator overnight. To a solution of dT-3 in trimethyl phosphate (5 mL), POCl (94.3 μL, 1.00 mmol) was added dropwise at 0 °C. The mixture was stirred at 0 °C for 2 h, then a well-vortexed mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (738 μL, 3.10 mmol) in anhydrous DMF (2 mL) was added. The mixture was stirred at room temperature for 1 h, then 0.1 M triethylammonium bicarbonate buffer (TEAB buffer, pH 8.5, 20 ml) was added and the mixture was stirred at room temperature overnight. The resulting mixture was concentrated under reduced pressure and the residue was diluted with 10 ml of water. The crude mixture was extracted with CHCl (2 × 10 mL) and the aqueous layer was concentrated under reduced pressure. The residue was then purified by anion exchange chromatography on DEAE-Sephadex A-25 using a gradient of TEAB (pH 8.5; 0.1-0.8M). Fractions containing the product (0.3M-0.4M) were collected and concentrated under reduced pressure. The residue was diluted with ddH2O and subjected to C18 HPLC (4% isocratic, 25 mM TEAB in acetonitrile: 25 mM TEAB in water). The product (retention time: 19 min) was collected and concentrated under reduced pressure to give dT-4 (14 mg, 6.5%) as a syrup. R f 0.28(1:2TEAB-ACN); 1 H NMR(400MHz,D2O):δ7.61(d,1H, 4 J=1.0Hz,H-6),6.19(dd,1H,J 1’,2’a =5.8Hz,J 1’,2’b =8.6Hz,H-1'),4.74(d,1H,J gem=8.8Hz,CH2N3),4.69(d,1H,J gem =8.8Hz,CH2N3),4.53-4.48(m,1H,H-3'),4.25-4.20(m,1H,H-4'),4.12-4.00(m,2H,H-5'a,H-5'b),2.37(ddd,1H,J 2’a,1’ =5.8Hz,J 2’a,3’ =2.0Hz,J gem =14.4Hz,H-2'a),2.25(ddd,1H,J 2’a,1’ =8.8Hz,J 2’a,3’ =5.9Hz,J gem =14.4Hz,H-2'b),1.78(d,3H, 4 J = 1.0Hz, CH3); 31 P NMR(121.4MHz,D2O):δ-10.95(bs,1P),-11.75(d,J=20.0Hz,1P),-23.36(bs,1P);HRMS(ESI)m / z[MH] - C 11 H 17 N5O 14 P3 - Calculated value 535.9990; measured value 535.9993. [ka] 6.10.2. Synthesis of L-3'-O-azidomethyl-dCTP: 6.10.2.1 Scheme 14. Synthesis of L-3'-O-N3-dCTP(dC-5) [ka]

[0229] Synthesis of dC-1: β-L-deoxycytidine (1 g, 4.40 mmol) was coevaporated with anhydrous pyridine (2×10 mL) and then dissolved in anhydrous pyridine (15 mL) under nitrogen. Trimethylsilyl chloride (TMSCl, 2.79 mL, 22.0 mmol) was slowly added and the mixture was stirred at room temperature for 1 h, then benzoyl chloride (2.56 mL, 22.0 mmol) was added and the solution was stirred at room temperature for another 24 h. After cooling to 0° C., water (10 mL) was added and the mixture was stirred at 0° C. for 20 min. Concentrated ammonia solution (15 mL) was then added and the solution was stirred for another 1 h while warming to room temperature. The solvent was evaporated under reduced pressure and the resulting crude product was purified by column chromatography (19:1, CH2Cl2-CH3OH) to give dC-1 (520 mg, 36%) as a white solid. R f 0.35 (1:19 CH3OH-CH2Cl2).

[0230] Synthesis of dC-2: To a solution of dC-1 (280 mg, 0.84 mmol) in anhydrous DMF (5.0 mL) with imidazole (172 mg, 2.53 mmol) and tert-butyldimethylsilyl chloride (204 mg, 1.35 mmol) at 0° C. The solution was stirred at 0° C. for 3 h under nitrogen. After completion of the reaction (TLC monitoring), cold water (10 mL) was added and extracted with EtOAc (3×20 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by column chromatography (19:1 to 9:1, CH2Cl2-CH3OH) to give dC-2 (280 mg, 74%) as a colorless oil. R f 0.32(1:19 CH3OH-CH2Cl2).

[0231] Synthesis of dC-3. To a stirred solution of dC-2 (240 mg, 0.538 mmol) in DMSO (6 ml) was added acetic acid (3 ml) and acetic anhydride (9 ml) at room temperature. The reaction mixture was stirred at room temperature for 72 h. Saturated NaHCO3 solution was added at 0° C., stirred for 30 min, and the aqueous layer was extracted with CH2Cl2 (2×30 ml). The combined organic extracts were dried over Na2SO4, filtered, and concentrated. The crude product was purified by flash column chromatography (1:1, hexane-EtOAc) to give dC-3 (163 mg, 60%) as a white powder. R f 0.22 (1:1 hexane-EtOAc); 1 H NMR (400MHz, CDCL3): δ8.42(d,1H,J=7.5Hz),7.92(d,2H,J=7.5Hz,ArH),7.67-7.58(m,1H,ArH),7.56-7.38(m,3H,ArH),6.29(app t,1H,J 1’,2’a =J 1’,2’b =6.0Hz,H-1'),4.69(d,1H,J gem =11.7Hz,CH2S),4.61(d,1H,J gem =11.7Hz,CH2S),4.50(app dt,1H,J 3’,2’a =J 3’,4’ =4.0Hz,J 3’,2’b =6.0Hz,H-3'),4.21-4.16(m,1H,H-4'),4.00(dd,1H,J 5’a,4’ =3.2Hz,J gem =11.8Hz,H-5'a),3.84(dd,1H,J 5’a,4’ =2.6Hz,J gem =11.8Hz,H-5'b),2.79(ddd,1H,J 2’a,1’ =6.0Hz,J 2’a,3’ =4.0Hz,J gem =13.7Hz,H-2'a),2.21-2.13(m,4H,H-2'b,SCH3),0.95(s,9H,(CH3)3CSi);0.15(s,3H,CH3Si),0.14(s,3H,CH3Si);HRMS(ESI)m / z [M+H] + C 24 H 36Calculated value of N3O5SSi: 506.2145; measured value: 506.2150.

[0232] Synthesis of dC-4. To a stirred solution of dC-3 (220 mg, 0.67 mmol) in dry CHCl (6 mL) was added cyclohexene (200 μL) and SOCl in CHCl (1.0 M, 0.6 mL). After stirring at 0 °C for 1.5 h, the volatiles were removed under reduced pressure. To a solution of the residue in dry DMF (3 mL) was added NaN (169 mg, 2.61 mmol) at room temperature for 3 h. The reaction mixture was dispersed in cold distilled water (30 mL) and extracted with EtOAc (2 × 30 mL). The combined organic extracts were dried over NaSO and concentrated under reduced pressure. The resulting residue was subjected to TBDMS removal reaction with 2 M HCl. The reaction was carried out at room temperature for 1-2 h. The mixture was neutralized with saturated NaHCO solution and diluted with EtOAc. The organic layer was dried over NaSO and concentrated under reduced pressure. The residue was purified by flash column chromatography (1:2, hexane-EtOAc to 100% EtOAc) to give dC-4 (73 mg, 56%) as a colorless oil. f 0.3 (1:4 hexane-EtOAc); 1 H NMR (400MHz, CDCL3): δ8.99(bs,1H,NH),8.32(d,1H,J=7.5Hz),7.87(d,2H,J=7.5Hz,ArH),7.65-7.53(m,2H),7.52-7.45(m,2H,ArH),6.18(app t,1H,J 1’,2’a =J 1’,2’b =6.3Hz,H-1'),4.79(d,1H,J gem =9.2Hz,CH2N3),4.68(d,1H,J gem =9.2Hz,CH2N3),4.52(app dt,1H,J 3’,2’a =J 3’,4’ =3.9Hz,J 3’,2’b =6.3Hz,H-3'),4.26-4.22(m,1H,H-4'),4.04(dd,1H,J 5’a,4’ =2.8Hz,J gem =12.0Hz,H-5'a),3.89(dd,1H,J 5’a,4’=2.8Hz,J gem =12.0Hz,H-5'b),3.45(bs,1H,5'-OH),2.68(ddd,1H,J 2’a,1’ =6.3Hz,J 2’a,3’ =3.9Hz,J gem =13.5Hz,H-2'a),2.44(app dt,1H,J 2’b,1’ =J 2’b,3’ =6.3Hz,J gem =13.5Hz,H-2'b);HRMS(ESI)m / z [M+H] + C 17 H 19 Calculated N6O5: 387.1417; Found: 387.1426.

[0233] Synthesis of dC-5: dC-4 (100 mg, 0.259 mmol) was dried over PO in a vacuum desiccator overnight. To a solution of dC-4 in trimethyl phosphate (5 mL), POCl (60.5 μL, 0.647 mmol) was added dropwise at 0 °C. The mixture was stirred at 0 °C for 2 h, then a well-vortexed mixture of tributylammonium pyrophosphate (355 mg, 0.647 mmol) and tributylamine (473.8 μL, 1.99 mmol) in anhydrous DMF (2 mL) was added. The mixture was stirred at room temperature for 1.5 h, then 0.1 M triethylammonium bicarbonate buffer (TEAB buffer, pH 8.5, 0.1 M, 15 ml) was added and the mixture was stirred at room temperature for 1 h. Concentrated ammonium hydroxide (10 mL) was then added to the mixture and stirred at room temperature overnight. The resulting mixture was concentrated under reduced pressure and the residue was diluted with 30 mL of water. The crude mixture was extracted with CHCl (2×20 mL) and the aqueous layer was concentrated under reduced pressure. The residue was then purified by anion exchange chromatography on DEAE-Sephadex A-25 using a gradient of TEAB (pH 8.5; 0.2-0.8 M). Fractions containing the product (0.3 M-0.4 M) were collected and concentrated under reduced pressure. The residue was diluted with ddH0 and subjected to C18 HPLC (2% isocratic, 25 mM TEAB in ACN: 25 mM TEAB in water). The product (retention time: 13.2 min) was collected and concentrated under reduced pressure to give dC-5 (18 mg, 13.4%) as a syrup. Rf 0.43 (1:2TEAB-ACN); 1 H NMR(400MHz,D2O):δ8.00-7.88(m,1H),6.23(dd,1H,J 1’,2’a =5.6Hz,J 1’,2’b =7.9Hz,H-1'),6.18-6.06(m,1H)4.85-4.74(m,2H,CH2N3),4.56-4.51(m,1H,H-3'),4.35-4.29(m,1H,H-4' ),4.18-4.09(m,2H,H-5'a,H-5'b),2.55-2.46(m,1H,H-2'a),2.31-2.19(m,1H,H-2'b);HRMS(ESI)m / z[MH] - C 10 H 16 N5O 13 P3 - Calculated value -520.9994; measured value 520.9990. 6.10.3.L-3'-O-azidomethyl-dATP synthesis: 6.10.3.1 Scheme 15. Synthesis of L-3'-O-N3-dATP (dA-5) [ka]

[0234] Testing Procedure: dA-1 synthesis: β-L-deoxyadenosine (2.0 g, 7.96 mmol) was coevaporated with anhydrous pyridine (2 times: 10 mL + 10 mL) and dissolved in anhydrous pyridine (20 mL). The resulting solution was cooled to 0 °C and chlorotrimethylsilane (TMSCl, 5.06 mL, 39.8 mmol) was added dropwise via syringe. The mixture was stirred at 0 °C for 1 h. Benzoyl chloride (4.62 mL, 39.8 mmol) was added dropwise via syringe to the solution and the ice bath was removed. After stirring at room temperature for 3 h, the flask was placed on an ice bath and concentrated ammonia solution (15 mL) was added. The solution was stirred at 0 °C for 30 min and then evaporated to dryness. The residue was dissolved in CH3OH and silica gel was added. The mixture was dried under reduced pressure. The residue was purified by column chromatography (49:1 to 19:1, CH2Cl2-CH3OH) to give dA-1 (2.12 g, 75%) as a white solid.

[0235] Synthesis of dA-2: To a solution of dA-1 (2.12 g, 5.98 mmol) in anhydrous DMF (30 mL) was added imidazole (608 mg, 8.95 mmol) and tert-butyldimethylsilyl chloride (989 mg, 6.56 mmol) at 0° C. under nitrogen. The reaction mixture was stirred at 0° C. for 3 h. After completion of the reaction, cold water (50 mL) was added and the mixture was extracted with EtOAc (3×50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated. The resulting residue was purified by column chromatography (1:2, hexane-EtOAc to 100% EtOAc) to give compound dA-2 (1.92 g, 69%) as a colorless oil.

[0236] Synthesis of dA-3: To a stirred solution of dA-2 (1.8 g; 3.83 mmol) in DMSO (24 ml) was added acetic acid (12 ml) and acetic anhydride (36 ml) at room temperature. After stirring at room temperature for 72 h. Saturated NaHCO3 solution was added to the solution at 0 °C and stirred for 30 min. The mixture was extracted with CH2Cl2 (2 x 100 ml). The combined organic layers were dried over Na2SO4, filtered and concentrated. The residue was purified by flash column chromatography (1:1, hexane-EtOAc) to give dA-3 (1.27 g, 63%) as a white foam. R f 0.28 (1:7 hexane-EtOAc); 1 H NMR (400MHz, CDCL3): δ9.28(bs,1H,NH),8.77(s,1H),8.33(s,1H),8.02(d,2H ,J=7.5Hz,ArH),7.62-7.55(m,1H,ArH),7.53-7.47(m,2H,ArH),6.51(dd,1H,J 1’,2’a =7.2Hz,J 1’,2’b =6.0Hz,H-1'),4.74-4.64(m,3H,CH2S,H-3'),4.25-4.18(m,1H,H-4'),3.89(dd,1H,J 5’a,4’ =4.4Hz,J gem =11.0Hz,H-5'a),3.82(dd,1H,J 5’a,4’ =3.3Hz,J gem =11.0Hz,H-5'b),2.79(ddd,1H,J 2’a,1’ =7.2Hz,J 2’a,3’ =6.0Hz,J gem =13.7Hz,H-2'a),2.63(ddd,1H,J 2’b,1’ =6.0Hz,J 2’b,3’ =3.0Hz,J gem =13.7Hz,H-2'b),2.18(s,3H,SCH3),0.91(s,9H,(CH3)3CSi);0.10(s,6H,(CH3)2Si);HRMS(ESI)m / z [M+H] + C 25 H 36 Calculated value of N5O4SSi: 530.2157; measured value: 530.2132. 6.10.4.L-3'-O-azidomethyl-dGTP Synthesis: 6.10.4.1 Scheme 16. Synthesis of L-3'-O-N3-dGTP (dG-6) [ka]

[0237] Testing Procedure: dG-1 synthesis: β-L-deoxyguanosine (2 g, 7.48 mmol) was coevaporated (2×12 mL), pyridine (30 mL) was added, and the suspension was cooled to 0° C. Trimethylsilyl chloride (4.8 mL, 37.4 mmol) was added dropwise via syringe. The ice bath was then removed and the mixture was stirred for 1 h. The solution was cooled to 0° C. and isobutyric anhydride (6.2 mL, 37.4 mmol) was added dropwise via syringe. The ice bath was removed and the resulting solution was stirred at room temperature for 3 h. After stirring for 3 h, the mixture was cooled to 0° C. and ice-cold water (5 mL) was added slowly and stirred for 15 min, followed by concentrated ammonia solution (10 mL) to a final concentration of 2.5 M ammonia. The mixture was stirred on the ice bath for 1 h and then evaporated to dryness. The residue was redissolved in CH3OH, silica gel was added, and concentrated to dryness. The mixture was concentrated to dryness, and the resulting residue was purified by column chromatography (19:1 to 9:1, CH2Cl2-CH3OH) to give dG-1 (1.85 g, 73%) as a white solid.

[0238] dG-2 synthesis: To a solution of dG-1 (1.8 g, 5.33 mmol) in anhydrous DMF (30 mL) was added imidazole (544 mg, 8.0 mmol) and tert-butyldimethylsilyl chloride (885 mg, 5.87 mmol) under nitrogen at 0° C. and warmed to room temperature. After stirring at room temperature for 8 h, ice-cold water was added to the solution and extracted with EtOAc (3×100 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated. The resulting residue was purified by column chromatography (19:1 to 9:1, CH2Cl2-CH3OH) to give compound dG-2 (1.9 g, 79%) as a white foam.

[0239] dG-3 synthesis: To a stirred solution of dG-2 (1.7 g; 3.76 mmol) in DMSO (12 ml) was added acetic acid (6 ml) and acetic anhydride (18 ml). After stirring at room temperature for 48 h, saturated NaHCO3 solution was added to the solution at 0° C., stirred for 30 min, and the aqueous layer was extracted with EtOAc (2×100 ml). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The crude product was purified by flash column chromatography (1:1 to 1:3, hexane-EtOAc) to give dG-3 (1.2 g; 65%) as a pale yellow foam. R f 0.26 (1:4 hexane-EtOAc); 1 H NMR(400MHz,CDCL3):δ12.1(s,1H,NH),9.40(s,1H,NH),7.98(s,1H),6.18(app t,1H,J 1’,2’a =J 1’,2’b =6.9Hz,H-1'),4.71-4.61(m,3H,CH2S,H-3'),4.15-4.09(m,1H,H-4'),3.78(d,2H,J=3.9Hz,H-5'a,H-5 'b),2.74(sep,1H,J=7.0Hz,COCH(CH3)2),2.57-2.46(m,2H,H-2'a,H-2'b),2.15(s,3H,SCH3),1.25(app t,6H,J=7.0Hz,COCH(CH3)2),0.89(s,9H,(CH3)3CSi);0.08(s,3H,(CH3)2Si),0.07(s,3H,(CH3)2Si);HRMS(ESI)m / z [M+H] + C 22 H 38 Calculated value of N5O5SSi: 512.2363; measured value: 512.2360.

[0240] Synthesis of dG-4. To a stirred solution of dG-3 (1.20 g; 2.35 mmol) in dry pyridine (25 ml) was added diphenylcarbamoyl chloride (814 mg; 3.52 mmol) and DIPEA (N,N-diisopropylethylamine) (1.23 ml; 7.03 mmol) at room temperature. After stirring at room temperature for 3 h, the solvent was removed under high vacuum. The residue was diluted with EtOAc and washed with 2 M HCl and saturated NaHCO3. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by flash column chromatography (1:1 to 1:3, hexane-EtOAc) to give dG-4 (1.5 g, 90%) as a foamy yellowish powder. R f 0.5 (1:1 hexane-EtOAc); 1 H NMR(400MHz,CDCL3):δ8.25(s,1H,NH),8.05(s,1H),7.50-7.33(m,8H,ArH),7.29-7.21(m,2H,ArH),6.41(app t,1H,J 1’,2’a =J 1’,2’b =6.7Hz,H-1'),4.76-4.66(m,3H,CH2S,H-3'),4.19-4.13(m,1H,H-4'),3.88(dd,1H,J 5’a,4’ =4.5Hz,J gem =11.1Hz,H-5'a),3.81(dd,1H,J 5’b,4’ =3.6Hz,J gem =11.1Hz,H-5'b),2.96(bs,1H,COCH(CH3)2),2.75(ddd,1H,J 2’b,1 =6.7Hz,J 2’a,3’ =7.2Hz,J gem =13.6Hz,H-2'a),2.57(ddd,1H,J 2’a,1 =6.7Hz,J 2’a,3’ =3.2Hz,J gem =13.6Hz,H-2'b),2.18(s,3H,SCH3),1.28(d,6H,J=6.8Hz,COCH(CH3)2),0.92(s ,9H,(CH3)3CSi);0.11(s,3H,(CH3)2Si),0.10(s,3H,(CH3)2Si);HRMS(ESI)m / z [M+H] + C 35H 47 Calculated N6O6SSi: 707.3047; measured: 707.3048.

[0241] Synthesis of dG-5: To a stirred solution of dG-4 (490 mg, 0.694 mmol) in dry CHCl (14 mL) was added cyclohexene (2.1 mL) and 1.0 M SOCl in CHCl (1.39 mL, 1.39 mmol) at 0 °C. After stirring at 0 °C for 1 h, the volatiles were removed under reduced pressure. To a solution of the residue in dry DMF (14 mL) was added NaN (271 mg, 4.61 mmol) at room temperature. After stirring at room temperature for 2 h, the reaction mixture was dispersed in distilled water (200 mL) and extracted with EtOAc (2 x 200 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure. To a solution of the residue in acetonitrile (14 mL) was added 2 M HCl (7 mL) at 0 °C. The reaction mixture was stirred for 30 min. The solution was diluted with EtOAc (100 mL) and washed with saturated NaHCO. The organic layer was washed with water (2 x 50 mL) and the organic layer was dried over NaSO, filtered and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (1:2 to 0:1, hexane-EtOAc) to give dG-4 as a colorless oil. f 0.32 (1:4 hexane-EtOAc); 1 H NMR(400MHz,CDCL3):δ8.38(s,1H,NH),8.08(s,1H),7.46-7.39(m,4H,ArH),7.37-7.32(m,4H,ArH),7.27-7.20(m,2H,ArH),6.22(app t,1H,J 1’,2’a =J 1’,2’b =6.6Hz,H-1'),5.04-4.98(m,1H,H-3'),4.77(s,2H,CH2N3),4.19-4.14(m,1H,H-4'),3.88(dd,1H,J 5’a,4’ =2.9Hz,J gem =12.6Hz,H-5'a),3.80(dd,1H,J 5’b,4’ =2.9Hz,J gem =12.6Hz,H-5'b),2.99(app dt,1H,J 2’b,1=J 2’a,3’ =6.6Hz,J gem =13.7Hz,H-2'a),2.79-2.66(m,1H,COCH(CH3)2),2.48(ddd,1H,J 2’a,1 =6.6Hz,J 2’a,3’ =3.8Hz,J gem =13.7Hz,H-2'b);HRMS(ESI)m / z [M+H] + C 28 H 30 Calculated value of N9O6: 588.2219; measured value: 588.2177.

[0242] 6.11. Example 11. (L) Complete synthesis of 3'-O-azidomethyl-dNTP labeled intermediate As described above in Example 3, the synthesis of L-3'-O-azidomethyl-dNTP-FL was designed and divided into three two parts: the synthesis of the linker (Scheme 17), the synthesis of the NH2-L-3'-O-azidomethyl-dNTP intermediate (Schemes 18-23), and then the coupling of the intermediate with a fluorophore.

[0243] The synthesis of the linker (Scheme 17) now gives compound 5, designated intermediate linker-5, which can be 1 Characterized by H NMR and HRMS. The first NH2-L-3'-O-azidomethyl-dNTP that we focused on is the synthesis of NH2-L-3'-O-azidomethyl-dUTP (Scheme 18). Currently, the synthesis of NH2-L-3'-O-azidomethyl-dUTP has been completed up to step 3, and intermediate "dUTP-FL-3" was obtained. The synthesis schemes of other L-3'-O-azidomethyl-dNTP-FL intermediates are also attached below (Schemes 19-21).

[0244] Considering the compatibility of fluorophore-attached dNTPs with 9°N DNA polymerase, we also designed the synthetic routes of NH2-(L)3'-O-N3-7-deaza-dGTP (Scheme 22) and NH2-(L)3'-O-N3-7-deaza-dATP (Scheme 23). The starting materials of nucleotide bases (6-chloro-7-deazaguanine and 6-chloro-7-deaza-7-iodopurine) and 2-deoxy sugars (1-chloro-3,5-di-Op-toluoyl-2-L-deoxyribofuranose) are commercially available. 6.11.1. Scheme 17. Synthesis of the Linker [ka]

[0245] Synthesis of Linker-1 To a solution of ethyl-3-hydroxybenzoate (3.32 g, 20 mmol) in anhydrous DMF (8 ML) was added potassium carbonate (5.53 g, 40 mmol), sodium iodide (1.2 g, 0.4 mmol), and 2-bromomethyl-1,3-dioxolane (8.3 mL, 80 mmol) at room temperature. The mixture was heated to 120° C. and stirred overnight. The mixture was warmed to room temperature and evaporated under reduced pressure. The residue was diluted with CHCl (250 mL) and washed with water. The aqueous layer was washed twice with CHCl (2×50 mL). The combined organic layers were dried over MgSO, filtered, and concentrated. The residue was purified by column chromatography (hexane-EtOAc, 19:1 to 2:1) to give linker-1 (4.6 g, 91%). R f 0.54 (100% CH2Cl2); 1 H NMR (400MHz, CDCL3): δ7.67(dt,1H,J=1.1,7.1Hz,ArH),7.61(dd,1H,J=1.5,2.6Hz,ArH),7.35(t,1H,J=7.8Hz,ArH),7.15(ddd,1H,J =1.0,2.7,8.6Hz,ArH),5.32(t,1H,J=4.0Hz,CH),4.38(q,2H,J=7.2Hz,OCH2CH3),4.12-3.96(m,6H,OCH2CH2O,ArOCH2),1.40(t,3H,Jgem = 7.2Hz, OCH2CH3);HRMS [ka]

[0246] Synthesis of Linker-2 To a solution of linker-1 (2.70 g, 10.7 mmol) in azidotrimethylsilane (1.55 mL, 11.8 mmol) was added tin(IV) chloride (80 μL) at room temperature. The mixture was stirred at room temperature for 2 h, then 2% aqueous methanol (10 mL) was added. The mixture was stirred at room temperature for 30 min, then concentrated under reduced pressure. The residue was co-evaporated with EtOH (2×30 mL), and the resulting residue was purified by column chromatography (hexane-EtOAc, 2:1 to 1:1) to give linker-2 (1.42 g, 45%). R f 0.32 (2:1 hexane-EtOAc); 1 H NMR(400MHz,CDCl3):δ7.70(dt,1H,J=1.1,7.6Hz,ArH),7.61(dd,1H,J=1.4,2.5Hz,ArH),7 .38(t,1H,J=7.8Hz,ArH),7.15(ddd,1H,J=0.9,2.5,8.1Hz,ArH),4.90(t,1H,J=5.3Hz,CHN 3),4.40(q,2H,J=7.2Hz,OCH2CH3),4.25(dd,1H,J=5.3,10.2Hz,ArOCH2),4.17(dd,1H,J=5 .3,10.2Hz,ArOCH2),4.06-4.00(m,1H,OCH2),3.91-3.73(m,3H,OCH2CH2OH),1.40(t,3H,J gem =7.2Hz,OCH2CH3);HRMS(ESI)m / z [MH] - C 15 H 18 Calculated value of N3O7: 352.1236; Found value: 352.1165 [ka]

[0247] Synthesis of Linker-3 To a solution of linker-2 (1.42 g, 4.8 mmol) in EtOH (5 mL) was added 4N NaOH (5 mL) at room temperature. The mixture was stirred at room temperature for 3 h, then the mixture was concentrated under reduced pressure, acidified with 2N HCl (20 mL), and extracted twice with CHCl (2×50 mL). The combined organic layers were dried over MgSO, filtered, and concentrated to give linker-3 (1.21 g, 94%). f 0.41(19:1 EtOAc-CH3OH); 1 H NMR (400MHz, CDCL3): δ7.78(dt,1H,J=1.1,7.6Hz,ArH),7.67(dd,1H,J=1.4,2.5Hz ,ArH),7.43(t,1H,J=7.8Hz,ArH),7.21(ddd,1H,J=0.9,2.5,8.1Hz,ArH),4.92(t,1 H,J=5.2Hz,CHN3),4.26(dd,1H,J=5.2,10.1Hz,ArOCH2),4.17(dd,1H,J=5.2,10.1 Hz,ArOCH2),4.08-4.01(m,1H,OCH2),3.89-3.77(m,3H,OCH2CH2OH);HRMS(ESI)m / z [MH] - C 11 H 12 Calculated value of N3O5: 266,0867; Measured value: 266.0794 [ka]

[0248] Synthesis of Linker-4 To a solution of linker-3 (320 mg, 1.57 mmol) in THF (5 mL) was added 60% NaH (188 mg, 4.71 mmol) at 0 °C. The mixture was stirred at 0 °C for 10 min, and then ethyl 2-bromoacetate (382 μL, 3.45 mmol) was added to the mixture. The mixture was warmed to room temperature and stirred for 4 h. After stirring for 4 h, cold water (50 mL) was poured into the reaction mixture and the resulting mixture was extracted with CHCl (50 mL). The organic layer was discarded and the aqueous layer was acidified by the addition of 2N HCl (25 mL). The resulting mixture was extracted with CHCl (2 × 50 mL) and the collected organic layers were dried over NaSO, filtered and concentrated. The resulting residue was purified by column chromatography (98:2, CH2Cl2-CH3OH) to give linker-4 (120 mg, 22%) as a pale yellow oil. f 0.67 (1:1:0.5 EtOAc-Hexane-AcOH); 1 H NMR(400MHz,CDCl3):δ7.75(app dt,1H,J=1.1,7.5Hz,ArH),7.64(dd,1H,J=1.4,2.5Hz,ArH),7.39(t,1H,J=7.8Hz,ArH),7.19(ddd,1H,J=0.8,2.6,8.2Hz,ArH),4.96(app t,1H,J=5.0Hz,CHN3),4.28-4.12(m,6H,ArOCH2,OCH2CH3,OCH2C=O),4.05(app dt,1H,J=4.1,11.4Hz,OCH2),3.97-3.87(m,1H,OCH2),3.82(appt,2H,J=4.8Hz,OCH2),1.29(t,3H,J=7.2Hz,OCH2CH3)HRMS(ESI)m / z [MH] - C 15 H 18 Calculated value of N3O7: 352.1236; Found value: 352.1165. [ka]

[0249] Synthesis of Linker-5 To a solution of linker-4 (120 mg, 0.339 mmol) in DMF (1 mL) was added DSC (104 mg, 0.407 mmol) and DMAP (49.8 mg, 0.407 mmol) at room temperature. The mixture was stirred at room temperature for 10 min, and then N-(2-aminoethyl)-2,2,2-trifluoroacetamide (78.5 mg, 0.407 mmol) and DIPEA (142 μL, 0.815 mmol) were added to the mixture at room temperature. The mixture was stirred at room temperature overnight and quenched with 1N Na2HPO4 solution (25 mL). The mixture was extracted with CH2Cl2 until no product was observed in the aqueous layer. The combined organic layers were dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by column chromatography (1:1, hexane-EtOAc) to give linker-5 (42 mg, 25%) as a colorless oil. f 0.62 (1:1:0.5 EtOAc-Hexane-AcOH); 1 H NMR (400MHz, CDCl3): δ8.25(bs,1H,NH),7.42-7.30(m,4H,ArH,NH),7.07(ddd,1H,J=0.9,2.5,8.0Hz,ArH),4.90(app t,1H,J=4.9Hz,CHN3),4.25-4.07(m,6H,ArOCH2,OCH2CH3,OCH2C=O),4.05-3.99(m,1H,OCH2),3.89-3.82(m,1H,OCH2),3 .79(appt,2H,J=4.2Hz,OCH2),3.70-3.62(m,2H,NCH2CH2N),3.61-3.54(m,2H,NCH2CH2N),1.28(t,3H,J=7.2Hz,OCH2CH3) [ka] 6.11.2. Synthesis of intermediate NH2-(L)3'-O-azidomethyl-dNTP: 6.11.2.1 Scheme 18. Synthesis of NH2-(L)3'-O-N3-dUTP [ka]

[0250] Testing Procedure: Synthesis of dUTP-FL-1: Beta-L-deoxyuridine (2.4 g, 10.51 mmol, 1.0 eq.) was suspended in methanol (30 mL) and stirred for 10 min. Then, iodine (8.0 g, 31.55 mmol, 3 eq.) and silver nitrate (5.3 g, 31.55 mmol, 3 eq.) were added and the reaction mixture was stirred at 40° C. for 3 h. After completion of the reaction, the reaction mixture was filtered and the filtrate was subjected to silica gel column chromatography using MeOH / CHCl 1:19 to 1:9 to obtain compound dUTP-FL-1 as a white solid (1.8 g, 48%). R f 0.65(9:1 DCM-MeOH), 1 HNMR(500MHz,DMSO-d6)δ11.63(s,1H),8.36(s,1H),6.05(t,J=6.5Hz,1H), 4.24-4.12(m,2H),3.79-3.69(m,2H),3.61-3.46(m,2H),2.15-1.96(m,2H). [ka]

[0251] Synthesis of dUTP-FL-2: Compound 1 (400 g, 1.12 mmol, 1.0 equiv.) was solubilized in anhydrous DMF (20 mL). To this mixture, imidazole (114.3 mg, 1.68 mmol, 1.5 equiv.) and tert-butyldimethylsilyl chloride (185.6 mg, 1.23 mmol, 1.1 equiv.) were added at 0° C. under nitrogen. The reaction mixture was stirred at room temperature for 12 h. Then, cold water was added and extracted with EtOAc (3×50 mL). The combined organic layers were dried over anhydrous Na2SO4, concentrated, and the resulting residue was subjected to column chromatography using 2-5% MeOH in CH2Cl2 as eluent to obtain compound dUTP-FL-2 as a foam-type white solid (348 mg, 63%). R f 0.45(19:1 DCM-MeOH) 1HNMR(500MHz,)δ8.38(s,1H),8.10(s,J=4.0Hz,1H),6.29(dd,J=8.1,5.6Hz,1 H),4.47(dd,J=5.4,3.1Hz,1H),4.08(q,J=2.3Hz,1H),3.86(ddd,J=40.5,11.4 ,2.5Hz,2H),2.41(ddd,J=13.4,5.6,2.1Hz,1H),2.09(ddd,J=13.6,8.1,5.7H z,2H),1.98(d,J=3.5Hz,1H),0.92(s,J=2.9Hz,9H),0.15(s,3H)-0.13(s,3H). [ka]

[0252] Synthesis of dUTP-FL-3: To a stirred solution of 2 (108 mg; 0.23 mmol) in DMSO (8 ml) was added acetic acid (4 ml) and acetic anhydride (12 ml). The reaction mixture was stirred at room temperature for 48 h. Saturated NaHCO3 solution (50 ml) was added at 0° C., stirred for 30 min, and the aqueous layer was extracted with EtOAc (3×100 ml). The combined organic extracts were dried over Na2SO4 and concentrated. The crude product was purified by flash column chromatography (ethyl acetate / hexane, 1:1 to 7:3) to give dUTP-FL-3 as a foamy white solid (106 mg, 87%). R f 0.85 (1:1 hexane-EtOAc) 1 H NMR(500MHz,)δ8.86(s),8.09(s),7.27(s),6.23(dd,J=8.3,5.5Hz),5.42-5.25(m),4.36(d,J=5.9Hz),4.17(d,J=2.1Hz),3.87(ddd,J=56.2,11 .5,2.3Hz),2.54(ddd,J=13.6,5.5,1.7Hz),2.12(s),2.04(ddd,J=14.0 ,8.3,5.9Hz),1.36-1.21(m,1H),0.95(s,9H),0.17(s,3H),0.16(s,3H). [ka] 6.12. Example 12. (L) Design of the synthesis of 3'-O-azidomethyl-7-deaza-dNTP-labeled 6.12.1.1 Scheme 19. Synthesis of NH2-L-3'-O-N3-dCTP [ka] 6.12.1.2 Scheme 20. Synthesis of NH2-L-3'-O-N3-dATP [ka] 6.12.1.3 Scheme 21. Synthesis of NH2-L-3'-O-N3-dGTP [ka] 6.12.1.4 Scheme 23. Synthesis of NH2-(L)3'-O-N3-7-Deaza-dATP [ka] Synthesis protocol for NH2-(L)3'-O-N3-7-Deaza-dATP: To a solution of 4-chloro-5-iodo-7H-pyrrolo[2.3-d]pyrimidine (1.0 g, 3.58 mmol) in CH3CH (60 mL) was added powdered KOH (85%, 0.5 g, 7.57 mmol) and TDA-1 (0.075 mL, 0.24 mmol) at room temperature. After stirring for 10 min, halogenose (1.7 g, 4.37 mmol) was introduced and stirring was continued for another 10 min. The insoluble material was filtered off and washed several times with hot acetone. The combined filtrates were evaporated to dryness. The residue was applied to flash chromatography (silica gel, column 5 x 15 cm, elution with perchloroethyl ether-EtOAc, 4:1). Evaporation of the combined fractions containing the product gave 4-chloro-7-[2-deoxy-3,5-di-O-(4-methylbenzoyl-β-L-erythro-pentofuranosyl)-5-iodo-7H-pyrrolo[2,3-d]pyrimidine as a colorless solid (2.02 g, 89%). A solution of the intermediate (1.8 g, 2.85 mmol) in NH3 / CH3OH (saturated at 0° C., 145 mL) was stirred at room temperature for 24 h and the solvent was evaporated. Flash chromatography (silica gel, column 4×16 cm, CHCl / CH3OH, 9:1) gave compound 7-deaza-dA-1 as a colorless solid (0.45 g, 40%). R f0.45 (CH2Cl2 / CH3OH, 9:1). To a stirred mixture of 7-deaza-dA-1 (1.00 g, 2.83 mmol) and imidazole (462 mg, 6.79 mmol) in anhydrous DMF (14.0 mL) was added tert-butyldimethylsilyl chloride (TBDMSCl) (510 mg, 3.28 mmol). The reaction mixture was stirred at room temperature for 20 h. After evaporation, the residue was purified by flash column chromatography (CH2Cl2-CH3OH, 20:1) to give 7-deaza-dA-2 as a white solid (1.18 g, 89%). To a stirred solution of compound 7-deaza-dA-2 in DMSO, acetic acid and acetic anhydride are added. The reaction mixture is stirred at room temperature for 48 h. Saturated NaHCO3 solution is added and the aqueous layer is extracted with CH2Cl2. The combined organic extracts are washed with saturated NaHCO3 solution and dried over Na2SO4. After concentration, the residue is purified by flash column chromatography (hexane / ethyl acetate, 1:1 to 1:4) to obtain compound 7-deaza-dA-3. To a stirred solution of compound 7-deaza-dA-3 in dry CHCl2 under nitrogen, cyclohexene and SO2Cl2 are added. The reaction mixture is stirred at 0°C for 2 h. The solvent is removed first under reduced pressure and then under high vacuum pump for 10 min. The residue is dissolved in dry DMF and reacted with NaN3 at room temperature for 3 h. The reaction mixture is dispersed in distilled water (50 ml) and extracted with CHCl2 (3 x 50 ml). The combined organic layers are dried over Na2SO4 and concentrated under reduced pressure. The residue is dissolved in MeOH and stirred with NHF at room temperature for 24 h. The solvent is removed under reduced pressure. The reaction mixture is concentrated under reduced pressure and partitioned between H2O and CH2Cl2. The organic layer is separated and dried over Na2SO4. After concentration, the crude product is purified by flash column chromatography (ethyl acetate / methanol, 100:0 to 98:2) to give compound 7-deaza-dA-4. Compound 7-deaza-dA-4 was dissolved in a 7M ammonia in methanol solution. The solution was stirred at 115-120 °C for 15 h. After evaporation, the residue was purified by flash column chromatography (CH2Cl2-CH3OH, 20:1) to give 7-deaza-dA-5.To a solution of 7-deaza-dA-5 in anhydrous DMF, tetrakis(triphenylphosphine)palladium(0) and CuI were added. The reaction mixture was stirred at room temperature for 10 minutes. Then, N-propargyltrifluoroacetamide and Et3N were added to the previous reaction mixture. The reaction was stirred at room temperature for 1.5 hours under the exclusion of iar and light. After evaporation, the residue was dissolved in ethyl acetate. The mixture was washed with saturated aqueous NaHCO3, NaCl, and dried over anhydrous Na2SO4. After evaporation, the residue was purified by flash column chromatography (CH2Cl2-CH3OH, 10:1) to obtain 7-deaza-dA-6. Compound 7-deaza-dA-6 and proton sponge were dried over P2O5 in a vacuum desiccator overnight and then dissolved in trimethyl phosphate. Freshly distilled POCl3 was then added dropwise at 0°C and the mixture was stirred at 0°C for 2 hours. A well-vortexed mixture of tributylammonium pyrophosphate and tributylamine in anhydrous DMF is then added in one portion at room temperature and stirred for 30 minutes. Triethylammonium bicarbonate solution (TEAB) (0.1M; pH 8.0) is then added and the mixture is stirred at room temperature for 1 hour. Concentrated NH4OH is then added and stirred at room temperature overnight. The resulting mixture is concentrated under vacuum and the residue is diluted with 5 ml of water. The crude mixture is then purified by anion exchange chromatography on DEAE-Sephadex A-25 at 4°C using a gradient of TEAB (pH 8.0; 0.1-1.0M). The crude product is further purified by reverse-phase HPLC to give (L) 3'-O-azidomethyl-7-deaza-dATP (compound 7-deaza-dA-7). 6.12.1.5 Scheme 25. (L) Synthesis of 3'-O-N3-7-Deaza-dATP-ROX [ka]

[0253] Azido-ROX compound (ROX-N3-linker). (2-{2-[3-(2-amino-ethylcarbamoyl)-phenoxy]-1-azido-ethoxy}-ethoxy)-acetic acid linker-6 (7.0 mg, 0.019 mmol), prepared according to the literature (Milton J, Ruediger S, Liu X (2006), US Patent Publication No. 20060160081), is dissolved in DMF (300 μl) and 1M aqueous NaHCO3 (100 μl). A solution of ROX NHS (N-hydroxysuccinimide) ester (Invitrogen) (0.013 mmol) in DMF (400 μl) is slowly added to the previous reaction mixture, which is then stirred at room temperature for 5 hours in the absence of light. The crude product is purified on a preparative silica gel TLC plate (CHCl3 / CH3OH, 1:4).

[0254] Compound L-3'-O-azidomethyl-7-deaza-dATP-ROX (L-3'-O-N3-7-deaza-dATP-ROX). To a stirred solution of ROX-N3-linker in dry DMF (2 ml), DSC (N,N'-disuccinimidyl carbonate) (3.4 mg, 13.2 μmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 μmol) are added. The reaction mixture is stirred for 2 h at room temperature. TLC shows that the ROX-N3-linker is completely converted to the compound ROX-N3-linker NHS ester, which is used directly to couple with L-amino-7-deaza-dATP (13 μmol) in NaHCO3 / Na2CO3 buffer (pH 8.7, 0.1 M) (300 μl). The reaction mixture is stirred for 3 h at room temperature with the exclusion of light. The reaction mixture is purified by preparative silica gel TLC plate (CHOH / CHCl, 1:1). The crude product is further purified by reverse phase HPLC to give L-3'-O-azidomethyl-dATP-ROX (L-3'-O-N-dATP-ROX). 6.12.1.6 Scheme 26. Synthesis of NH2-(L)3'-O-N3-deaza-dGTP [ka]

[0255] Protocol for the synthesis of NH2-(L)3'-O-N3-7-deaza-dGTP. To a suspension of powdered KOH (85%, 1.15 g, 17.42 mmol) and TDA-1 (0.2 mL, 0.63 mmol) in CH3CN (60 mL) was added 2-amino-4-chloro-7H-pyrrolo[2.3-d]pyrimidine (842 mg, 4.99 mmol) at room temperature. After stirring for 5 min, halogenose (2.53 g, 6.51 mmol) was introduced within 15 min and stirring was continued for 30 min. Insoluble material was filtered off and washed several times with CH3CN. The combined filtrates were evaporated to dryness. The residue was applied to flash chromatography (silica gel, column 6 x 12 cm, elution with CHCl2). The combined fractions containing the product were evaporated to give a colorless solid (2.21 g, 85%). A solution of the intermediate (1.04 g, 2.00 mmol) in 0.5 M NaOCH3 / CH3OH (60 mL) was stirred under reflux for 3 h. The mixture was neutralized with AcOH and evaporated. The residue was applied to flash chromatography (silica gel, CHCl2 / CH3OH, 95:5) to give compound 7-deaza-dG-1 as a colorless solid (400 mg, 71%). The stirred solution of 7-deoxy-dG-1 was coevaporated with anhydrous pyridine (3×4 mL) and then dissolved in anhydrous pyridine (2 mL). The resulting solution was protected from moisture (dry tube), purged with argon, and placed on ice. To the ice-cold solution, TMS-Cl (4.51 mL, 58.4 mmol, 8.2 equiv.) was added dropwise via syringe. The ice bath is then removed and the mixture is stirred for 2 h. The solution is cooled on ice and isobutyric chloride (0.29 mL, 15.69 mmol, 2.2 equiv) is added dropwise via syringe and the ice bath is removed. After stirring at room temperature for another 2 h, the reaction is again placed on ice and ice-cold water (20 mL) is added slowly followed after 15 min by concentrated ammonia solution (1.5 mL) to give a final concentration of ammonia of 2.5 M. The mixture is kept on ice for 30 min and then evaporated to dryness. The residue is coevaporated with toluene (3 x 5 mL) to remove traces of water, resuspended in MeOH and filtered to remove the precipitate.The filtrate is then concentrated, dissolved in a small amount of MeOH, absorbed onto silica gel, and purified by column chromatography (DCM / MeOH 95:5 to 91:9 (v / v)) to give compound 7-deaza-dG-2, which is coevaporated with dry pyridine three times, dried under high vacuum, and cooled in an ice bath for 2 cm. 3The mixture is dissolved in dry N,N-dimethylformamide. Imidazole (419 mg, 6.15 mmol) is added and the mixture is stirred at 0° C. for 15 min and at room temperature for 15 min. Then, 241 mg of tert-butyldimethlsiliyl chloride (1.59 mmol) is added and the solution is stirred at 60° C. for another 2 h. The mixture is diluted with dichloromethane, washed with brine, dried over sodium sulfate and evaporated. The crude product is purified as a white foam (compound 7-deaza-dG-3) by column chromatography on silica gel (methanol:dichloromethane 0:100 to 2:98). To a vigorously stirred solution of 7-deaza-dG-3 in anhydrous DMF, NIS was added. The reaction mixture was stirred at room temperature for 22 h and then most of the solvent was removed under vacuum. Diethyl ether and saturated NaHCO3 were added. The organic layer was washed with saturated NaCl and dried over Na2SO4. After evaporation, the residue was purified by flash column chromatography to obtain 7-deaza-dG-4. To a stirred solution of compound 7-deaza-dG-4 in DMSO (12 ml), acetic acid (5.5 ml) and acetic anhydride (17.6 ml) were added. The reaction mixture was stirred at room temperature for 48 h. Saturated NaHCO3 solution (100 ml) was added and the aqueous layer was extracted with CHCl2 (3×100 ml). The combined organic extracts were washed with saturated NaHCO3 solution (100 ml) and dried over NaSO4. After concentration, the residue was purified by flash column chromatography to obtain compound 7-deaza-dG-5. To a stirred solution of compound 7-deaza-dG-5 in dry CHCl2 under nitrogen, cyclohexene and SO2Cl2 were added. The reaction mixture was stirred at 0° C. for 2 h. The solvent is removed first under reduced pressure and then under high vacuum pump for 10 min. The residue is dissolved in dry DMF and reacted with NaN3 at room temperature for 3 h. The reaction mixture is dispersed in distilled water (50 ml) and extracted with CHCl2 (3 x 50 ml). The combined organic layers are dried over NaSO4 and concentrated under reduced pressure. The residue is purified by flash column chromatography (ethyl acetate / methanol, 100:0 to 98:2) to give compound 7-deaza-dG-6.To a solution of 7-deaza-dG-6 in anhydrous DMF, tetrakis(triphenylphosphine)palladium(0) and CuI were added. The reaction mixture was stirred at room temperature for 10 min. Then, N-propargyltrifluoroacetamide and Et3N were added to the previous reaction mixture. The reaction was stirred at room temperature for 1.5 h under the exclusion of iar and light. After evaporation, the residue was dissolved in ethyl acetate. The mixture was washed with saturated aqueous NaHCO3, NaCl, and dried over anhydrous Na2SO4. After evaporation, the residue was purified by flash column chromatography to obtain 7-deaza-dG-7. To a stirred solution of 7-deaza-dG-7 in anhydrous CH3CN, NaI and chlorotrimethylsilane were added. The reaction was stirred at room temperature for 1 h and then at 50 °C for 12 h. The solvent was evaporated and the residue was dissolved in THF. Tetrabutylammonium fluoride (TBAF) in THF solution was added and the reaction was stirred at room temperature for 1 h. The solvent was evaporated and the residue was dissolved in EtOAc. The solution was washed with saturated aqueous NaCl and dried over anhydrous Na2SO4. After evaporation of the solvent, the residue was purified by flash column chromatography (EtOAc-CH3OH) to obtain 7-deaza-dG-8. Compound 7-deaza-dG-8 and proton sponge were dried over P2O5 in a vacuum desiccator overnight and then dissolved in trimethyl phosphate. Freshly distilled POCl3 was then added dropwise at 0°C and the mixture was stirred at 0°C for 2 h. Subsequently, a well-vortexed mixture of tributylammonium pyrophosphate and tributylamine in anhydrous DMF was added in one portion at room temperature and stirred for 30 min. Triethylammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0) was then added and the mixture was stirred at room temperature for 1 h. Concentrated NH4OH was then added and stirred at room temperature overnight. The resulting mixture is concentrated under vacuum and the residue is diluted with 5 ml of water. The crude mixture is then purified by anion exchange chromatography on DEAE-Sephadex A-25 at 4° C. using a gradient of TEAB (pH 8.0; 0.1-1.0 M). The crude product is further purified by reverse-phase HPLC to give (L) 3′-O-azidomethyl-7-deaza-dATP (compound 7-deaza-dG-9). 6.12.1.7 Scheme 27. (L) 3'-O-N3-7-Deaza-dGTP-Cy5 [ka]

[0256] Azide-Cy5 compound (Cy5-N3-Linker). (2-{2-[3-(2-amino-ethylcarbamoyl)-phenoxy]-1-azido-ethoxy}-ethoxy)-acetic acid linker-6 (7.0 mg, 0.019 mmol), prepared according to the literature (Milton J, Ruediger S, Liu X (2006), US Patent Publication No. 20060160081), is dissolved in DMF (300 μl) and 1M NaHCO3 aqueous solution (100 μl). A solution of Cy5 NHS (N-hydroxysuccinimide) ester (Invitrogen) (0.013 mmol) in DMF (400 μl) is slowly added to the previous reaction mixture, which is then stirred at room temperature for 5 hours in the absence of light. The crude product is purified on a preparative silica gel TLC plate (CHCl3 / CH3OH, 1:4). Compound L-3'-O-azidomethyl-7-deaza-dGTP-Cy5 (L-3'-O-N3-7-deaza-dGTP-Cy5). To a stirred solution of Cy5-N3-linker in dry DMF (2 ml), DSC (N,N'-disuccinimidyl carbonate) (3.4 mg, 13.2 μmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 μmol) are added. The reaction mixture is stirred at room temperature for 2 hours. TLC shows that Cy5-N3-linker is completely converted to the compound Cy5-N3-linker NHS ester, which is used directly to couple with L-amino-7-deaza-dGTP (13 μmol) in NaHCO3 / Na2CO3 buffer (pH 8.7, 0.1 M) (300 μl). The reaction mixture is stirred at room temperature for 3 hours in the absence of light. The reaction mixture is purified by preparative silica gel TLC plate (CHOH / CHCl, 1:1). The crude product is further purified by reverse phase HPLC to obtain L-3'-O-azidomethyl-dGTP-Cy5 (L-3'-O-N3-dGTP-Cy5).

[0257] 6.13. Example 13. Chemical synthesis of (D)-type 9°N DNA polymerase via solid-phase peptide synthesis and native chemical ligation

[0258] Figure 1 provides the amino acid sequence of the (D)-type mutant 9°N DNA polymerase (candidate #1-1), where all amino acids are D-type amino acids. Diamonds = mutations introduced for NCL; Circles = NCL sites (start of the "2nd" strand); Squares = potential substitution sites for isoleucine; Triangles = mutations introduced into modified nucleotides. [Table 2-1] [Table 2-2]

[0259] The 9°N DNA polymerase was split into two fragments (466-aa 9°NN fragment and 310-aa 9°NC fragment) at the split site between K466 and M467. The synthesis of the 466 aa 9°NN fragment was designed as nine synthetic peptides (D-9°NN-1 to D-9°NN-9) via solid-phase peptide synthesis ligated at specific cysteine ​​residues shown in Figure 2B (Figure 2A). Similarly, the 310-aa 9°C fragment was split into six synthetic peptides (Figure 3A), which were synthesized by solid-phase peptide synthesis and then ligated at specific cysteine ​​residues shown in Figure 3B.

[0260] Experimental Method: Materials: 2-Chlorotrityl chloride resin (loading = 0.98 mmol g -1Fmoc-D-amino acids, D-4-thiazolidine carboxylic acid, hydrazine hydrate, ethyl cyanoglyoxylate-2-oxime (Oxyma), N,N'-diisopropylcarbodiimide (DIC), trifluoroacetic acid, N,N-dimethylformamide (DMF), dichloromethane, piperidine, thioanisole, triisopropylsilane, 1,2-ethanedithiol, and trifluoroacetic acid for peptide synthesis were purchased commercially from Chempep, Sigma-aldrich, Alfa aesar, TCI, etc. Reagents for NCL reaction, namely guanidine hydrochloride (Gn·HCl), Na2HPO4·12H2O, NaH2PO4·2H2O, sodium nitrite (NaNO2), sodium hydroxide (NaOH), sodium 2-mercaptoethanesulfonate hydrochloride, 4-mercaptophenylacetic acid (MPAA), tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl), DL-1,4-dithiothreitol (DTT), 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044), glutathione (reduced form), and palladium chloride (PdCl2) were commercially purchased from Sigma-aldrich, Alfa aesar, TCI, Duksan, etc.

[0261] Fmoc-based SPPS. All peptides were synthesized by Fmoc-based SPPS on a Liberty Blue automated microwave peptide synthesizer (CEM) and a PurePep® Chorus automated peptide synthesizer. All peptide hydrazides were synthesized on hydrazine-2-chlorotrityl chloride resin. For each peptide hydrazide, the first residue was attached to the hydrazine-2-chlorotrityl chloride resin by a double coupling method using 5 equivalents of amino acid, 10 equivalents of DIC, and 5 equivalents of Oxymapure. All resins were swollen in DMF for 30 minutes before coupling. The Fmoc groups of the assembled amino acids were removed by treatment with 20% piperidine and 0.1 M Oxyma in DMF at 85°C. Coupling of amino acids other than Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH was carried out at 85°C for 2 min using 5 equivalents of amino acid, 5 equivalents of Oxymapure, and 10 equivalents of DIC. Coupling reactions for Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH were carried out at 50°C for 10 min to avoid side reactions at high temperatures. Trifluoroacetylthiazolidine-4-carboxylic acid-OH was coupled overnight at room temperature using 5 equivalents of Oxymapure, and 10 equivalents of DIC. For peptides with more than 20 amino acids, a double coupling strategy was used. After completion of peptide chain assembly, the peptides were cleaved from the resin using H2O / thioanisole / triisopropylsilane / 1,2-ethanedithiol / trifluoroacetic acid (0.5 / 0.5 / 0.5 / 0.25 / 8.25) (vol / vol). The cleavage reaction took 2.5 h under stirring at 27°C. Cold ether was added to precipitate the crude peptide. After centrifugation, the supernatant was discarded and the precipitate was washed twice with ether. The crude peptide was dissolved in CH3CN / H2O, analyzed by RP-HPLC and purified by semi-preparative HPLC. The collected peptide fractions were analyzed by electrospray ionization mass spectrometry (ESI-MS).

[0262] Native Chemical Ligation (NCL). The C-terminal peptide hydrazide segment was dissolved in acidified ligation buffer (aqueous solution of 6 M Gn·HCl and 0.1 M NaH2PO4, pH 3.0). The mixture was cooled in an ice-salt bath (-15 °C) and 10 equivalents of NaNO2 in acidified ligation buffer (pH 3.0) were added. The activated reaction was kept under stirring in the ice-salt bath for 20 min, after which 40 equivalents of MPAA and 1 equivalent of N-terminal cysteine ​​peptide in ligation buffer were added to adjust the pH of the solution to 6.6-6.8 at room temperature. After overnight reaction, the system was diluted twice by adding 150 mM TCEP in ligation buffer (pH adjusted to 7.0) and the reaction was kept under stirring at room temperature for 30 min. Finally, the ligation product was analyzed by HPLC and purified by semi-preparative HPLC. The purified ligation fraction was analyzed by ESI-MS.

[0263] Tfa-D-Thz-OH synthesis: [ka]

[0264] To a solution of D-4-thiazolidine carboxylic acid (1 g, 7.509 mmol) suspended in MeOH (40 mL), triethylamine (2.62 mL, 18.772 mmol) was added and the suspension was stirred for 10 min. Ethyl trifluoroacetate (0.98 mL, 8.2599 mmol) was then added dropwise and the mixture was stirred at room temperature for 48 h. The mixture was then concentrated and subjected to silica gel chromatography using MeOH / dichloromethane (1:5) to give Tfa-(D)-Thz-OH, i.e., (S)-3-(2,2,2-trifluoroacetyl)thiazolidine-4-carboxylic acid) as a pale yellow oil (0.9 g, 3.92 mmol, 52.3%) (Figure 4A). 1H NMR (500 MHz, CDCl3): δ (ppm) 9.35 (s, 1H), 5.09 (dd, J = 6.9, 4.2 Hz, 1H), 4.86-4.77 (m, 1H, rotat), 4.75-4.61 (m, 1H, rotat), 3.52-3.28 (m, 2H, rotat) (Figure 4B).

[0265] Fmoc-based SPPS (Figure 5): 1) 102 mg of 2-Cl-(Trt)-Cl resin (0.98 mmol g -1 ) into a peptide synthesis vessel. 2) Add 5 ml of DMF to the resin through the top of the reaction vessel, gently mix for 10 seconds, then drain. 3) Repeat step 2 two more times. 4) Add 5ml of DCM to the resin, gently mix for 10 seconds, then drain. 5) Repeat step 4 two more times. 6) Repeat step 2 three more times. 7) The resin is swelled in 4 ml of 50% (v / v) DMF / DCM for 30 minutes and then drained. 8) For hydrazination, add 4 ml of 5% (v / v) NH2NH2·H2O to the resin. Gently agitate the mixture in a thermostatic shaker at 30 °C for 30 min, then drain the solution by vacuum filtration. 9) Add 4 ml of DMF to the resin, gently mix for 10 seconds, then drain. 10) Repeat step 8. 11) Wash the resin by repeating steps 2-6 twice. 12) Add 4 ml of 5% (v / v) MeOH / DMF to the resin and gently agitate for 10 minutes before draining into any unreacted sites on the resin to cap. 13) Repeat steps 2 to 6 to thoroughly wash the resin. 14) Add 4 ml of DCM to the resin, gently mix for 10 seconds, then drain. Use the resin directly in the next coupling step. 15) For storage, dry the resin under high vacuum for 2 hours and store the dried 2-Cl-(Trt)-NHNH2 resin at -20°C under argon gas for up to 2 weeks.

[0266] Solid-phase peptide synthesis of pentamer D-9°NN-6: Tfa-Thz-AVYE-NHNH2 (FIG. 6A). Pentamer D-9°NN-6 was synthesized by PurePep® Chorus automated peptide synthesizer according to the conditions described in the experimental method. Pentamer D-9°NN-6 synthesized by this process was analyzed by HPLC chromatogram, and the results are shown in FIG. 6B-FIG. 6C.

[0267] 11-mer D-9°N-6: CVFGKPKEKVY-NHNH2: Solid-phase peptide synthesis of 11-mer D-9°N-6 was performed on a PurePep® Chorus automated peptide synthesizer according to the conditions described in the experimental method. The 11-mer D-9°NN-6 synthesized by this process was analyzed by HPLC chromatogram, and the results are shown in Figures 7B to 7D.

[0268] Solid-phase peptide synthesis of 24-mer D-9°NN-6: CEEIAQAWE SGEGLERVAR YSMED-NHNH2 (SEQ ID NO: 13) (FIG. 8A). 24-mer D-9°NN-6 was synthesized on a PurePep® Chorus automated peptide synthesizer according to the conditions described in the experimental methods. 24-mer D-9°NN-6 synthesized by this process was analyzed by HPLC chromatogram, and the results are shown in FIG. 8B-FIG. 8C.

[0269] 9-mer D-9°NC-3: CDTDGLHAT-NHNH2 (SEQ ID NO: 14) (FIG. 9A), solid-phase peptide synthesis of 9-mer D-9°NC-3 was performed on a Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) according to the conditions described in the experimental method. The 9-mer D-9°NC-3 synthesized by this process was analyzed by HPLC chromatogram, and the results are shown in FIG. 9B-FIG. 9C.

[0270] Solid-phase peptide synthesis of 33-mer customized D-9°NC-1: MKATVDPLEK KLLDYRQRLI KILANSFYGYYGY-NHNH2. The 33-mer D-9°NC-1 synthesized by this process was analyzed by HPLC chromatogram, and the results are shown in Figure 10A-10B.

[0271] Solid-phase peptide synthesis of 39-mer customized D-9°NC-2: CKARWY-C(Acm)-KE-C(Acm)AESVTAWGRE YIEMVIRELE EKFGFKVLY-NHNH2. The 39-mer D-9°NC-2 synthesized by this process was analyzed by HPLC chromatogram, and the results are shown in Figure 11A-B.

[0272] Solid-phase peptide synthesis of 21-mer D-9°NC-3: TFA-Thz-DTDGLHATIPGADAETVKKK-NHNH2 (SEQ ID NO: 15). 21-mer D-9oN-C-3 was synthesized by Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) according to the conditions described in the experimental method. The product was observed as 21-mer TFA-deprotected D-9°NC-3, as shown in Figure 12B and Figure 12C.

[0273] Cys35-mer D-9°NC-3: CKEFLKYINP KLPGLLELEY EGFYVRGFFV TKKKY-NHNH2. Solid phase peptide synthesis of (SEQ ID NO: 16). o Solid phase peptide synthesis of NC-3 was performed using a Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) according to the conditions described in the experimental procedures. The product was observed to have the expected mass, as shown in Figures 13B-13D.

[0274] Solid phase peptide synthesis of 56-mer D-9° NC-3: CDTDGLHATIPGADAETVKKKAKEFLKYINPKLPGLLELEYEGFYVRGFFVTKKKY-NHNH2 (SEQ ID NO: 17) (FIG. 14A).o Solid phase peptide synthesis of NC-3 was performed using a Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) according to the conditions described in the experimental procedures. The product was observed to have the expected mass, as shown in Figure 14B.

[0275] 35-mer D-9° NC-3: AKEFLKYINP KLPGLLELEY EGFYVRGFFV TKKKY-NHNH2 (SEQ ID NO: 18) (Figure 15A). o Solid phase peptide synthesis of NC-3 was performed using a Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) according to the conditions described in the experimental procedures. The product was observed to have the expected mass, as shown in Figure 15B.

[0276] Native Chemical Ligation (NCL): Preparation of reagents: NaOH, 1M and 6M. Dissolve 40mg or 240mg NaOH in 1ml deionized H2O. HCl, 6M. Mix 1ml of 12M HCl with 1ml deionized H2O. Two different phosphate solutions (0.1M) containing 6M Gn·HCl (pH 3.0-3.1 and pH 5.7-6.0, respectively). For a 10ml solution, mix 156mg NaH2PO4·2H2O and 5.74g Gn·HCl in a 10ml volumetric flask and adjust to final volume with deionized H2O. Adjust pH to 3.0-3.1 with 6M NaOH and 6M HCl. Filter the solution by using a 13mm x 0.22μm microporous membrane filter. The filtered solution can be stored at 4°C for at least one month. Na2HPO4·12H2O (0.1M) containing 6M Gn·HCl (pH 5.7-6.0) was prepared similarly. NaNO2, 0.5M. Dissolve 17mg NaNO2 in 0.5ml deionized H2O. TCEP, 0.1M. Dissolve 58mg TCEP·HCl in 1ml 0.2M phosphate solution (pH 3.0) containing 6M guanidine hydrochloride (Gn·HCl). Adjust the pH to 6.0-7.0 and filter the solution by using a 13mm x 0.22μm microporous membrane filter. VA-044, 0.1M. Weigh 9.7mg VA-044 into a 2ml Eppendorf reaction tube and add 0.3ml 0.2M phosphate solution (pH 6.9-7.0) containing 6M Gn·HCl. The VA-044 is completely dissolved by vortexing and using an ultrasonic cleaning bath.

[0277] 16-mer D-9 o NN-6: Synthesis of Tfa-Thz-AVYECVFGKPKEKVY-NHNH2 (SEQ ID NO: 19 and SEQ ID NO: 20) (FIG. 16A). o The preparation process of NN-6 is shown in Figure 16B. o The HPLC chromatogram analysis of NN-6 is shown in FIG. 16C.

[0278] Synthesis of 72-mer D-9oN-C-7: MKATVDPLEKKLLDYRQRLIKILANSFYGYYGYCKARWY-C(Acm)-KE-C(Acm)AESVTAWGRE YIEMVIRELE EKFGFKVLY-NHNH2 (SEQ ID NO: 21, 22, 23) (Figure 17A). The preparation process of 72-mer D-9oN-C-7 is shown in Figure 17B. Figure 17C is an analytical HPLC chromatogram (λ=214 nm) of peak conversion during the progression of the NCL reaction. Figure 17D is an ESI-MS of 72-mer D-9°NC-7 with MPAA attachment, and Figure 17E shows the ligated attached MPAA.

[0279] 7. Equivalents and Incorporation by Reference While the present invention has been particularly shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

[0280] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes. 8. Arrays [Table 3-1] [Table 3-2] [Table 3-3]

Claims

1. a. a pentose sugar selected from (3R,4S)-3,4,5-trihydroxypentanal and (4R)-4,5-dihydroxypentanal, in which the H of the 5' hydroxyl is replaced by one or more phosphate groups and the H of the 3' hydroxyl group, if present, is optionally replaced by a cleavable protecting group; b. a nitrogenous base; c. optionally a cleavable label comprising a cleavable linker and a label; A nucleotide or nucleoside comprising: A nucleotide or nucleoside in which at least one cleavable protecting group and a cleavable label are present.

2. 2. The nucleotide of claim 1, wherein the cleavable label is linked to the nitrogenous base, the 3'O, or the 5' phosphate group.

3. Formula I: 【Chemistry 75】 having a structure according to 2. The nucleotide of claim 1, wherein the base is a nitrogenous base and R' is a cleavable protecting group.

4. Formula II 【Chemical Formula 76】 having a structure according to where base is a nitrogenous base; R′ is a cleavable protecting group or H; R ２ The label is a cleavable linker R ２ and a cleavable label comprising the label.

5. Formula III: 【Chemical 77】 having a structure according to wherein the base is a nitrogenous base; R ２ The label is a cleavable linker R ２ and a cleavable label comprising the label.

6. The label is 【Chemical 78】 The nucleotide of claim 1, 2, 4, or 5, selected from the group consisting of:

7. The nucleotide of any one of claims 1 to 4, wherein the cleavable protecting group is selected from allyl, dimethyl disulfide, nitrobenzyl, and azide protecting groups.

8. The cleavable protecting group is 【Chemical 79】 The nucleotide of claim 7, selected from:

9. 6. The nucleotide of claim 1, 2, 4, or 5, wherein the cleavable linker is photocleavable, cleaved by contact with a water-soluble phosphine, or cleaved by a water-soluble transition metal-containing catalyst.

10. 10. The nucleotide of claim 9, wherein the cleavable linker comprises an allyl or azide group.

11. The cleavable linker is 【Chemistry 80】 The nucleotide of claim [0124],

12. 【Chemical 81】 is selected from In the formula, R′ is 【Chemistry 82】 The nucleotide of claim 3, selected from: 【Request 13】 【Chemical 83-1】 【Chemistry 83-2】 is selected from In the formula, R′ is 【Chemistry 84】 is selected from R ２ teeth, 【Chemistry 85】 The nucleotide of claim 4, comprising: 【Request 14】 【Chemical 86-1】 【Chemistry 86-2】 is selected from In the formula, R ２ teeth, 【Chemistry 87】 The nucleotide of claim 5 . 【Request 15】 【Chemical 88-1】 【Hua 88-2】 is selected from wherein R′ is: 【Chemistry 89】 The nucleotide of claim 4, selected from: 【Request 16】 【Chemical 90-1】 【Chemistry 90-2】 【Chemistry 90-3】 is selected from In the formula, R′ is 【Chemistry 91】 The nucleotide of claim 5, selected from:

17. A mirror image nucleic acid polymerase comprising a sequence having at least 90% sequence identity to SEQ ID NO:1, the polymerase comprising D-amino acids.

18. 20. The polymerase of claim 17, which is comprised of D-amino acids.

19. 19. The polymerase of claim 17 or 18, comprising a sequence having at least 95% sequence identity with SEQ ID NO:

1.

20. 20. The polymerase of claim 19, comprising a sequence having at least 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:

1.

21. 21. The polymerase of any one of claims 17 to 20, comprising one or more modifications at one or more amino acid positions selected from E276, K317, N424, and S651 compared to SEQ ID NO:

1.

22. 22. The polymerase of claim 21, comprising one or more substitutions selected from E276A, K317G, N424A, and S651A compared to SEQ ID NO:

1.

23. 23. The polymerase of claim 22, comprising the following substitutions compared to SEQ ID NO:1: E276A, K317G, N424A, and S651A.

24. 24. The polymerase of any one of claims 17 to 23, comprising one or more modifications at one or more amino acid sites selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733, and I744, compared to SEQ ID NO:

1.

25. 25. The polymerase of claim 24, comprising one or more substitutions of Ile to Ala, Val, Leu, or Tyr at one or more amino acid sites selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733, and I744, compared to SEQ ID NO:

1.

26. 26. The polymerase of claim 25, comprising one or more substitutions selected from I80V, I127V, I171A, I176V, I191V, I228V, I256V, I264A, I268L, I400V, I597V, I610V, I618A, I630L, I642V, I715Y, I733V, and I744V compared to SEQ ID NO:

1.

27. 27. The polymerase of claim 26, comprising the following substitutions compared to SEQ ID NO:1: I80V, I127V, I171A, I176V, I191V, I228V, I256V, I264A, I268L, I400V, I597V, I610V, I618A, I630L, I642V, I715Y, I733V, and I744V.

28. 28. The polymerase of any one of claims 17 to 27, comprising one or more modifications at one or more amino acid sites selected from M129, I130, G131, D141, E143, L408, Y409, P410, A485, T514, and I521, compared to SEQ ID NO:

1.

29. 29. The polymerase of claim 28, comprising one or more modifications at one or more amino acid sites selected from D141, E143, Y409, and A485, compared to SEQ ID NO:

1.

30. 30. The polymerase of claim 29, comprising one or more substitutions selected from D141A, E143A, Y409V, and A485L compared to SEQ ID NO:

1.

31. 31. The polymerase of claim 30, comprising the following substitutions compared to SEQ ID NO:1: D141A, E143A, Y409V, and A485L.

32. 29. The polymerase of claim 28, comprising one or more modifications at one or more amino acid sites selected from D141, E143, L408, Y409, P410, A485, T514, and I521, compared to SEQ ID NO:

1.

33. 33. The polymerase of claim 32, comprising one or more substitutions selected from D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L compared to SEQ ID NO:

1.

34. 34. The polymerase of claim 33, comprising the following substitutions compared to SEQ ID NO:1: D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L.

35. 29. The polymerase of claim 28, comprising one or more modifications selected from the following substitutions compared to SEQ ID NO:1: M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S, I521L, or the addition of a D between I130 and G131.

36. 36. The polymerase of claim 35, comprising the substitutions M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L, and the addition of D between I130 and G131, compared to SEQ ID NO:

1.

37. 37. The polymerase of any one of claims 17 to 36, comprising a sequence selected from SEQ ID NOs: 2 to 7.

38. 38. The polymerase of claim 37, consisting of a sequence selected from SEQ ID NOs: 2 to 7.

39. A method for replicating an L-polynucleotide, comprising inducing replication of the L-polymerase by incubating a mixture containing (i) the L-polynucleotide, (ii) an L-primer, (iii) L-dNTP, (iv) a polymerase according to any one of claims 1 to 38, and (v) a buffer.

40. The method according to claim 39, wherein said L-polynucleotide is DNA or RNA.

41. The method according to any one of claims 39 to 40, wherein the mixture comprises L-dATP, L-dGTP, L-dCTP, and L-dTTP.

42. The buffer is 50 mM Tris-HCl, pH 7.5, 20 mM MgCl ２ 1 mM DTT, and 50 mM KCl.

43. The method of any one of claims 39 to 42, wherein the incubation step comprises PCR.

44. A method for sequencing an L-polynucleotide, comprising: a. (i) the L-polynucleotide, (ii) the L-primer, (iii) the L-3'-O-R'-dNTP-R ２ - obtaining a replication product by incubating a mixture comprising a label, (iv) a polymerase according to any one of claims 17 to 38, and (v) a buffer; b. The L-3'-O-R'-dNTP-R incorporated into the replication product ２ - detecting a signal from the label; and c. The L-3'-O-R'-dNTP-R incorporated into the replication product ２ The R′ group and R ２ Induce cleavage of the group A method comprising:

45. 45. The method of claim 44, wherein the cycle is repeated at least 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 times.

46. The L-3'-O-R'-dNTP-R ２ - the label is L-3'-O-R'-dATP-R ２ -label, L-3'-O-R'-dTTP-R ２ -label, L-3'-O-R'-dGTP-R ２ -label, and L-3'-O-R'-dCTP-R ２ - labels, each label being different.

47. The L-3'-O-R'-dNTP-R ２ - The method according to any one of claims 44 to 46, wherein the label has a structure according to formula II as defined in any one of claims 4, 6 to 13 and 15.

48. The L-3'-O-R'-dNTP-R ２ The method according to claim 47, wherein the label is a nucleotide according to claim 13 or 15.

49. The method according to any one of claims 44 to 48, wherein the signal is a fluorescent signal.

50. A method for sequencing an L-polynucleotide, comprising: a. (i) the L-polynucleotide, (ii) an L-primer, (iii) an L-dNTP, and (iv) an L-ddNTP-R ２ -label or L-3'-O-R'-dNTP-R ２ - Obtaining a replication product by incubating a mixture comprising a label, (v) a polymerase according to any one of claims 17 to 38, and (vi) a buffer; b. separating the replication products; c. The L-ddNTP-R incorporated into the replication product ２ -label or L-3'-O-R'-dNTP-R ２ - detecting a signal from the label; A method comprising:

51. The method of claim 50, wherein the L-dNTPs include L-dATP, L-dTTP, L-dGTP, and L-dCTP.

52. The L-ddNTP-R ２ - The label is L-ddATP-R ２ -label, L-ddTTP-R ２ -label, L-ddGTP-R ２ -label, and L-ddCTP-R ２ - labels, each label being different.

53. The L-ddNTP-R ２ - A method according to any one of claims 50 to 52, wherein the label has formula III as defined in any one of claims 5, 6, 9 to 11.

54. The L-ddNTP-R ２ The method of claim 53, wherein the label is a nucleotide according to claim 14 or 16.

55. The L-3'-O-R'-dNTP-R ２ - the label is L-3'-O-R'-dATP-R ２ -label, L-3'-O-R'-dTTP-R ２ -label, L-3'-O-R'-dGTP-R ２ -label, and L-3'-O-R'-dCTP-R ２ - labels, each label being different.

56. The L-3'-O-R'-dNTP-R ２ - the method according to any one of claims 50 to 51 and 55, wherein the label has formula II as defined in any one of claims 4, 6 to 13 and 15.

57. The L-3'-O-R'-dNTP-R ２ The method of claim 56, wherein the label is a nucleotide according to claim 13 or 15.

58. The method of any one of claims 50 to 57, wherein the signal is a fluorescent signal.

59. 59. The method of any one of claims 50 to 58, wherein the incubation step comprises PCR.

60. 60. The method of any one of claims 50 to 59, wherein the separating step comprises separating the replication products by size.

61. A method for sequencing an L-polynucleotide, comprising: a. (i) the L-polynucleotide, (ii) the L-primer, (iii) L-3'-O-R'-dNTP, (iv) L-ddNTP-R ２ -label or L-dNTP-R ２ - obtaining a replication product by incubating a mixture comprising a label, (v) a polymerase according to any one of claims 17 to 38, and (vi) a buffer; b. The L-ddNTP-R incorporated into the replication product ２ -label or L-3'-O-R'-dNTP-R ２ - detecting a signal from the label; and c. i) the R' group of the L-3'-O-R'-dNTP incorporated into the replication product and the L-ddNTP-R ２ -R on the sign ２ or ii) the R' group of the L-3'-O-R'-dNTP incorporated into the replication product, L-3'-O-R'-dNTP-R ２ -Labels R' and R ２ Induce cleavage of the group A method comprising:

62. 62. The method of claim 61 , wherein the cycle is repeated at least 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 times.

63. The method of any one of claims 61 to 62, wherein the L-3'-O-R'-dNTPs include L-3'-O-R'-dATP, L-3'-O-R'-dTTP, L-3'-O-R'-dGTP, and L-3'-O-R'-dCTP.

64. 64. The method of any one of claims 61 to 63, wherein the L-3'-O-R'-dNTP has a structure according to formula I as defined in any one of claims 3, 7, and 8.

65. The method of claim 64, wherein the L-3'-O-R'-dNTP is a nucleotide according to claim 12.

66. The L-ddNTP-R ２ - The label is L-ddATP-R ２ -label, L-ddTTP-R ２ -label, L-ddGTP-R ２ -label, and L-ddCTP-R ２ - labels, each label being different.

67. The L-ddNTP-R ２ - The method according to any one of claims 61 to 66, wherein the label has a structure according to formula III as defined in any one of claims 5, 6, and 9 to 11.

68. The L-ddNTP-R ２ The method of claim 64, wherein the label is a nucleotide according to claim 14 or 16.

69. The L-3'-O-R'-dNTP-R ２ - the label is L-3'-O-R'-dATP-R ２ -label, L-3'-O-R'-dTTP-R ２ -label, L-3'-O-R'-dGTP-R ２ -label, and L-3'-O-R'-dCTP-R ２ - labels, each label being different.

70. The L-3'-O-R'-dNTP-R ２ - The method according to any one of claims 58 to 63 and 69, wherein the label has a structure according to formula II as defined in any one of claims 4, 6 to 13 and 15.

71. The L-3'-O-R'-dNTP-R ２ The method according to claim 70, wherein the label is a nucleotide according to claim 13 or 15.

72. The method of any one of claims 61 to 71, wherein the signal is a fluorescent signal.

73. A kit for the replication of L-polynucleotides comprising (i) a polymerase according to any one of claims 17 to 38, and (ii) optionally a buffer.

74. 74. The kit of claim 73, further comprising L-dNTPs.

75. The kit of claim 74, wherein the L-dNTP comprises L-dATP, L-dGTP, L-dCTP, L-dTTP, or L-UTP.

76. L-3'-O-R'-dNTP-R ２ The kit according to any one of claims 73 to 75, further comprising a label.

77. The L-3'-O-R'-dNTP-R ２ - the label is L-3'-O-R'-dATP-R ２ -label, L-3'-O-R'-dTTP-R ２ -label, L-3'-O-R'-dGTP-R ２ -label, L-3'-O-R'-dCTP-R ２ -label, or L-3'-O-R'-dUTP-R ２ The kit of claim 76, further comprising a label.

78. The L-3'-O-R'-dNTP-R ２ - A kit according to any one of claims 76 to 77, wherein the label has formula II as defined in any one of claims 4 to 11.

79. The L-3'-O-R'-dNTP-R ２ The kit according to claim 78, wherein the label is a nucleotide according to claim 13 or 15.

80. L-ddNTP-R ２ The kit according to any one of claims 73 to 79, further comprising a label.

81. The L-ddNTP-R ２ - The label is L-ddATP-R ２ -label, L-ddTTP-R ２ -label, L-ddGTP-R ２ -label, L-ddCTP-R ２ -label, or L-ddUTP-R ２ The kit of claim 80, further comprising a label.

82. The L-ddNTP-R ２ - a kit according to any one of claims 76 to 81, wherein the label has formula III as defined in any one of claims 5, 6 and 9 to 11.

83. The L-ddNTP-R ２ The kit according to claim 82, wherein the label is a nucleotide according to claim 14 or 16.

84. The kit according to any one of claims 73 to 83, further comprising L-3'-O-R'-dNTPs.

85. 85. The kit of claim 84, wherein the L-3'-O-R'-dNTPs include L-3'-O-R'-dATP, L-3'-O-R'-dTTP, L-3'-O-R'-dGTP, and L-3'-O-R'-dCTP.

86. The kit according to any one of claims 84 to 85, wherein the L-3'-O-R'-dNTP has formula I as defined in claim 3 or 12.

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