Methods of sequencing l-polynucleotides

Abstract

The present disclosure provides novel methods of amplifying and sequencing L-type polynucleotides. Further provided include L-type nucleotides and D-type enzymes that can be used in the methods. Methods of making L-type nucleotides and D-type enzymes are also provided.

Description

[0001] 1. Sequence Listing

[0002] This application contains a Sequence Listing.

[0003] 2. Background

[0004] 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 changes.

[0005] 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) that can be characterized as L or D based on optical activity. However, except for glycine, which does not have chirality, most amino acids present in nature have L-type. Nucleic acids have their chiral centers on their backbones. Unlike amino acids, nucleic acids are mainly D-type in nature. Due to chiral specificity, biomolecules can interact and use other molecules or substrates only when they have a certain chirality. For example, L-type polymerases can use D-type nucleotides instead of L-type nucleotides to synthesize polynucleotides.

[0006] Recently, 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 the advantage of such slower biodegradation in vivo. However, the development of mirror-image biomolecules and systems faces significant obstacles due to the lack of efficient and reliable technologies for sequencing L-polynucleotides.

[0007] 3. Overview

[0008] The present disclosure provides a novel method for sequencing L-type polynucleotides. Compositions for the sequencing method are also provided, such as novel L-type nucleotides and D-type enzymes. It is expected that the sequencing method can achieve high-throughput sequencing of L-polynucleotides.

[0009] Thus, in a first aspect, the present disclosure provides a mirror nucleotide comprising:

[0010] a. A pentose selected from (3R,4S)-3,4,5-trihydroxypentanal and (4R)-4,5-dihydroxypentanal; wherein the H of the 5' hydroxyl group is replaced by one or more phosphate groups; and wherein the H of the 3' hydroxyl group (if present) is optionally replaced by a cleavable protecting group;

[0011] b. Nitrogenous bases, and

[0012] c. optionally a cleavable label comprising a cleavable linker and a label;

[0013] At least one of a cleavable protecting group and a cleavable label is present.

[0014] In certain embodiments, the cleavable tag is attached to a nitrogenous base, a 3'0, or a 5' phosphate group.

[0015] In certain embodiments, the mirror nucleotide has a structure according to Formula I:

[0016]

[0017] wherein the base is a nitrogenous base and R' is a cleavable protecting group.

[0018] In certain embodiments, the mirror nucleotide has a structure according to Formula II

[0019]

[0020] wherein the base is a nitrogenous base; R' is a cleavable protecting group or H; R 2 — The label contains a cleavable linker R 2 and a labeled cleavable tag.

[0021] In certain embodiments, the mirror nucleotide has a structure according to Formula III:

[0022]

[0023] The base is a nitrogenous base; R 2 — The label contains a cleavable linker R 2 and a labeled cleavable tag.

[0024] In certain embodiments of the mirror nucleotide of Formula II or III, the label is selected from the group consisting of:

[0025]

[0026] In certain embodiments of the mirror nucleotide of Formula I or II, the cleavable protecting group is selected from allyl, dimethyldisulfide, nitrobenzyl, and azido protecting groups.

[0027] In certain embodiments of the mirror nucleotide of Formula I or II, the cleavable protecting group is selected from:

[0028]

[0029] In certain embodiments of the mirror nucleotide 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.

[0030] In certain embodiments of the mirror nucleotide of Formula II or III, the cleavable linker comprises an allyl group, a disulfide group, or an azide group.

[0031] In certain embodiments of the mirror nucleotide of Formula II or III, wherein the cleavable linker comprises:

[0032]

[0033] In certain embodiments of the mirror nucleotide of Formula I, the compound has a structure selected from:

[0034]

[0035] In certain embodiments, R' comprises a methyldisulfide, allyl, azide, or nitrobenzyl moiety.

[0036] In certain embodiments, R' is selected from:

[0037]

[0038] In certain embodiments of the mirror nucleotide of Formula II, the compound has a structure selected from:

[0039]

[0040]

[0041] In certain embodiments, R' comprises a methyldisulfide, allyl, azide, or nitrobenzyl moiety.

[0042] In certain embodiments, R' is selected from:

[0043] H. and

[0044] In certain embodiments, R 2 Include:

[0045]

[0046]

[0047] In certain embodiments of the mirror nucleotide of Formula III, the compound has a structure selected from:

[0048]

[0049]

[0050] In certain embodiments, R 2 Include:

[0051]

[0052] In certain embodiments of the mirror nucleotide of Formula II, the compound has a structure selected from:

[0053]

[0054]

[0055] Wherein R' is selected from:

[0056] H. In certain embodiments of the mirror nucleotide of Formula III, the compound has a structure selected from:

[0057]

[0058]

[0059] In certain embodiments, R' comprises a methyldisulfide, allyl, azide, or nitrobenzyl moiety.

[0060] In certain embodiments, R' is selected from:

[0061] H.

[0062] In a second aspect, the present disclosure provides a mirror 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.

[0063] In certain embodiments, the polymerase consists of D-amino acids.

[0064] 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.

[0065] In certain embodiments, the polymerase comprises one or more modifications at one or more amino acid positions selected from E276, K317, N424, and S651 compared to SEQ ID NO: 1.

[0066] In certain embodiments, the polymerase comprises one or more substitutions selected from E276A, K317G, N424A, and S651A compared to SEQ ID NO: 1.

[0067] In certain embodiments, the polymerase comprises E276A, K317G, N424A, and S651A substitutions compared to SEQ ID NO:1.

[0068] In certain embodiments, the polymerase comprises one or more modifications at one or more amino acid positions 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.

[0069] In certain embodiments, the polymerase comprises one or more substitutions from Ile to Ala, Val, Leu or Tyr at one or more amino acid positions selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733 and I744 as compared to SEQ ID NO: 1.

[0070] 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.

[0071] In certain embodiments, the polymerase comprises substitutions of 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.

[0072] In certain embodiments, the polymerase comprises one or more modifications at one or more amino acid positions selected from M129, I130, G131, D141, E143, L408, Y409, P410, A485, T514, and I521 compared to SEQ ID NO: 1.

[0073] In certain embodiments, the polymerase comprises one or more modifications at one or more amino acid positions selected from D141, E143, Y409, and A485 compared to SEQ ID NO: 1.

[0074] In certain embodiments, the polymerase comprises one or more substitutions selected from the group consisting of D141A, E143A, Y409V, and A485L compared to SEQ ID NO: 1.

[0075] In certain embodiments, the polymerase comprises substitutions of D141A, E143A, Y409V, and A485L compared to SEQ ID NO:1.

[0076] In certain embodiments, the polymerase comprises one or more modifications at one or more amino acid positions selected from D141, E143, L408, Y409, P410, A485, T514, and I521 compared to SEQ ID NO: 1.

[0077] In certain embodiments, the polymerase comprises one or more substitutions selected from the group consisting of D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L compared to SEQ ID NO: 1.

[0078] In certain embodiments, the polymerase comprises substitutions of D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L compared to SEQ ID NO: 1.

[0079] In certain embodiments, the polymerase comprises one or more substitution modifications selected from M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S, I521L, or an addition of D between I130 and G131 compared to SEQ ID NO: 1.

[0080] In certain embodiments, the polymerase comprises substitutions of M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L, and an addition of D between I130 and G131, compared to SEQ ID NO: 1.

[0081] In certain embodiments, the polymerase comprises a sequence selected from SEQ ID NOs: 2-7.

[0082] In certain embodiments, the polymerase consists of a sequence selected from SEQ ID NOs: 2-7.

[0083] In a third aspect of the present disclosure, a method for replicating an L-polynucleotide is provided, the method comprising the following steps: incubating a mixture comprising (i) an L-polynucleotide, (ii) an L-primer, (iii) L-dNTPs, (iv) a polymerase as described in any one of claims 1 to 38, and (v) a buffer, thereby inducing the replication of the L-polymerase.

[0084] In certain embodiments, the L-polynucleotide is DNA or RNA.

[0085] In certain embodiments, the mixture comprises L-dATP, L-dGTP, L-dCTP, and L-dTTP.

[0086] In certain embodiments, the buffer comprises 50 mM Tris-HCl, pH 7.5, 20 mM MgCl 2 , 1 mM DTT and 50 mM KCl.

[0087] In certain embodiments, the incubating step comprises PCR.

[0088] In a fourth aspect of the present disclosure, there is provided a method for sequencing an L-polynucleotide, the method comprising the following cycles:

[0089] a. comprising (i) L-polynucleotide, (ii) L-primer, (iii) L-3'-O-R'-dNTP-R 2 - incubating a mixture of (iv) a label, a polymerase according to any one of claims 17 to 38, and (v) a buffer, thereby obtaining a replication product;

[0090] b. Detection of L-3'-O-R'-dNTP-R from incorporated replication products 2 - a signal for the marker; and

[0091] c. Induce L-3'-O-R'-dNTP-R incorporated into the replication product 2 - labeled R' groups and R 2 Cleavage of the group.

[0092] In certain embodiments, the cycle is repeated at least 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 times.

[0093] In certain embodiments, L-3'-O-R'-dNTP-R 2 - Label contains L-3'-O-R'-dATP-R 2 -label, L-3'-O-R'-dTTP-R 2 -label, L-3'-O-R'-dGTP-R2 -Tag and L-3'-O-R'-dCTP-R 2 -labels, wherein each label is different. In certain embodiments, L-3'-O-R'-dNTP-R 2 - The 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.

[0094] In a fifth aspect of the present disclosure, there is provided a method for sequencing an L-polynucleotide, the method comprising the following steps:

[0095] a. comprising (i) L-polynucleotide, (ii) L-primer, (iii) L-dNTP, (iv) L-ddNTP-R 2 -label or L-3'-O-R'-dNTP-R 2 - incubating a mixture of (v) a label, a polymerase according to any one of claims 17 to 38, and (vi) a buffer, thereby obtaining a replication product;

[0096] b. separating the replication products; and

[0097] c. Detection of L-ddNTP-R from incorporated replication products 2 -label or L-3'-O-R'-dNTP-R 2 -Marked signal.

[0098] In certain embodiments, the L-dNTP comprises L-dATP, L-dTTP, L-dGTP, and L-dCTP. 2 - Tag contains L-ddATP-R 2 -Marker, L-ddTTP-R 2 -label, L-ddGTP-R 2 -Marker and L-ddCTP-R 2 - tags, where each tag is different.

[0099] In certain embodiments, L-ddNTP-R 2 - The tag has Formula III as defined in Section 5.2.2.2.1. In certain embodiments, L-3'-O-R'-dNTP-R 2 - Label contains L-3'-O-R'-dATP-R 2 -label, L-3'-O-R'-dTTP-R 2 -label, L-3'-O-R'-dGTP-R 2 -Tag and L-3'-O-R'-dCTP-R 2-labels, wherein each label is different. In certain embodiments, L-3'-O-R'-dNTP-R 2 - The label has Formula II as defined in Section 5.2.2.1.2. In certain of these embodiments, the signal is a fluorescent signal.

[0100] In certain of these embodiments, the incubating step comprises PCR.In certain of these embodiments, the separating step comprises separating the replicated products by size.

[0101] In a sixth aspect of the present disclosure, there is provided a method for sequencing an L-polynucleotide, the method comprising the following cycles:

[0102] a. comprising (i) L-polynucleotide, (ii) L-primer, (iii) L-3'-O-R'-dNTP, (iv) L-ddNTPs-R 2 -labeled or L-dNTPs-R 2 - incubating a mixture of (v) a label, a polymerase according to any one of claims 17 to 38, and (vi) a buffer, thereby obtaining a replication product;

[0103] b. Detection of L-ddNTP-R from incorporated replication products 2 -label or L-3'-O-R'-dNTP-R 2 - a signal for the marker; and

[0104] c. Inducing cleavage of: i) the R' group of L-3'-O-R'-dNTP and L-ddNTPs-R 2 -Marked R 2 or ii) the R' group of the L-3'-O-R'-dNTP incorporated into the replication product, the L-3'-O-R'-dNTP-R 2 - Labeled R' and R 2 Group.

[0105] In certain embodiments, the cycle is repeated at least 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 times.

[0106] In certain 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.

[0107] 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.

[0108] In certain embodiments, L-ddNTP-R 2 - Tag contains L-ddATP-R 2 -Marker, L-ddTTP-R 2 -label, L-ddGTP-R 2 -Marker and L-ddCTP-R 2 - tags, where each tag is different.

[0109] In certain embodiments, L-ddNTP-R 2 - The tag has a structure according to Formula III as defined in Section 5.2.2.2.1. In certain embodiments, L-3'-O-R'-dNTP-R 2 - Label contains L-3'-O-R'-dATP-R 2 -label, L-3'-O-R'-dTTP-R 2 -label, L-3'-O-R'-dGTP-R 2 -Tag and L-3'-O-R'-dCTP-R 2 -labels, wherein each label is different. In certain embodiments, L-3'-O-R'-dNTP-R 2 - The 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.

[0110] In a sixth aspect of the present disclosure, there is provided a kit for replicating an L-polynucleotide, the kit comprising (i) the polymerase according to any one of the preceding embodiments and (ii) optionally a buffer.

[0111] 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.

[0112] In certain embodiments, the kit comprises L-3'-O-R'-dNTP-R 2 -labeling. In certain embodiments, the kit comprises L-3'-O-R'-dNTP-R 2 -labeled, containing 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-R2 -labeling. In certain embodiments, the kit comprises L-3'-O-R'-dNTP-R 2 - a marker having Formula II as defined in Section 5.2.2.1.2.

[0113] In certain embodiments, the kit comprises L-ddNTP-R 2 -labeling. In certain embodiments, the kit comprises L-ddNTP-R 2 - Label, containing L-ddATP-R 2 -Marker, L-ddTTP-R 2 -label, L-ddGTP-R 2 -Marker, L-ddCTP-R 2 - Marker or L-ddUTP-R 2 -label. In certain embodiments, L-ddNTP-R 2 - The marker has Formula III as defined in Section 5.2.2.2.1.

[0114] In certain embodiments, the kit comprises L-3'-O-R'-dNTPs. In certain embodiments, the L-3'-O-R'-dNTPs comprise L-3'-O-R'-dATP, L-3'-O-R'-dTTP, L-3'-O-R'-dGTP, and L-3'-O-R'-dCTP. In certain embodiments, the L-3'-O-R'-dNTPs have Formula I as defined in Section 5.2.2.1.1. 4. Brief description of the accompanying drawings

[0116] These and other features, aspects and advantages of the present invention will become better understood with reference to the following description and accompanying drawings, in which:

[0117] Figure 1 The amino acid sequence of the (D)-type mutant 9°N DNA polymerase (candidate #1-1) is provided (SEQ ID NO:8). All amino acids are D-type amino acids. Diamonds = mutations introduced for NCL; circles = NCL site (start of the 'second' chain); squares = potential substitution sites for isoleucine; triangles = mutations introduced for modified nucleotides.

[0118] Figure 2A The sequence of D-type 9°N DNA polymerase mutant 1-I, 9°NN fragment (SEQ ID NO:9) is shown, with the sequences of nine synthetic peptides presented in separate rows. Figure 2B The synthetic pathway for D-type 9°N DNA polymerase mutant 1-I, 9°NN fragment is shown.

[0119] Figure 3A The sequence of D-type 9°N DNA polymerase mutant 1-I, 9°NC fragment (SEQ ID NO: 10) is shown, with the sequences of six synthetic peptides presented in separate rows. Figure 3B The synthetic pathway for D-type 9°N DNA polymerase mutant 1-I, 9°NC fragment is shown.

[0120] Figure 4A A TLC stain of Tfa-(D)-Thz-OH (ie, (S)-3-(2,2,2-trifluoroacetyl)thiazolidine-4-carboxylic acid) is provided. Figure 4B Provided is Tfa-(D)-Thz-OH (i.e., (S)-3-(2,2,2-trifluoroacetyl)thiazolidine-4-carboxylic acid) 1 HNMR (500 MHz, CDCl 3 )result.

[0121] Figure 5 As detailed in Example 13, 2-Cl-(Trt)-NHNH 2 synthesis.

[0122] Fig. 6A It is the peptide sequence of D-9°oN-N-6@5-mer (SEQ ID NO: 11). Figure 6B An analytical HPLC chromatogram (λ=214 nm) of crude D-9°NN-6@5-mer is provided. Column: Welch C4. Gradient: 100%-80% A in B from 0-20 min, 80%-50% A in B from 20-40 min, 50%-20% A in B from 40-60 min, and 20%-0% A in B from 60-70 min. Where A=H 2 O (0.1% TFA) and B=CH 3 CN (0.1% TFA). Figure 6C An analytical HPLC chromatogram of pure D-9°NN-6@5-mer is provided. Gradient: 2 95%-5% CH in O(0.1% TFA) 3 CN (0.1% TFA) for 30 min. Purification conditions: Column: Welch C4 semi-preparative column. Gradient: 100%-80% A in B from 1-20 min, 80%-50% A in B from 20-40 min, 50%-20% A in B from 40-50 min, and 20%-0% A in B from 50-55 min. Where A = H 2 O (0.1% TFA) and B=CH 3 CN (0.1% TFA).

[0123] Fig. 7A is the peptide sequence of D-9°NN-6@11-mer (SEQ ID NO: 12); Figure 7B An analytical HPLC chromatogram (λ=214 nm) of crude D-9°oN-N-6@11-mer is provided. Column: Welch C4. Gradient: at H 2 95%-5% CH in O(0.1% TFA) 3 CN (0.1% TFA), for 30 min; Figure 7C An analytical HPLC chromatogram of pure D-Pfu-9°oN-N-6@11-mer is provided. Gradient: 2 95%-5% CH in O(0.1% TFA) 3 CN (0.1% TFA) for 30 min. Purification conditions: Column: Welch C4 semi-preparative column. Gradient: 100%-80% A in B from 1-20 min, 80%-50% A in B from 20-40 min, 50%-20% A in B from 40-50 min, and 20%-0% A in B from 50-55 min. Where A = H 2 O (0.1% TFA) and B=CH 3 CN (0.1% TFA); Fig.7D The MALDI-TOF mass spectrum of D-9°oN-N-6@11-mer is provided.

[0124] Fig. 8A is the peptide sequence of D-9°oN-N-6@24-mer (SEQ ID NO: 13); Figure 8B An analytical HPLC chromatogram (λ=214 nm) of crude D-9°oN-N-6@24-mer is provided. Column: Welch C4. Gradient: 100%-80% A in B from 0-20 min, 80%-50% A in B from 20-40 min, 50%-20% A in B from 40-60 min, and 20%-0% A in B from 60-70 min. Where A=H 2 O (0.1% TFA) and B=CH 3 CN (0.1% TFA); Figure 8C An analytical HPLC chromatogram of pure D-Pfu-9°oN-N-6@5-mer is provided. Gradient: 2 95%-5% CH in O(0.1% TFA) 3CN (0.1% TFA) for 30 min. Purification conditions: Column: Welch C4 semi-preparative column. Gradient: 100%-80% A in B from 1-20 min, 80%-50% A in B from 20-40 min, 50%-20% A in B from 40-50 min, and 20%-0% A in B from 50-55 min. Where A = H 2 O (0.1% TFA) and B = CH 3 CN (0.1% TFA).

[0125] Fig. 9A is the peptide sequence of D-9°NC-3@9-mer (SEQ ID NO: 14); Fig. 9B An analytical HPLC chromatogram (λ=214 nm) of crude D-9°NC-3@9-mer is provided. Column: Welch C4. Gradient: 100%-80% A in B from 0-20 min, 80%-50% A in B from 20-40 min, 50%-20% A in B from 40-60 min, and 20%-0% A in B from 60-70 min. Where A=H 2 O (0.1% TFA) and B=CH 3 CN (0.1% TFA). Purification conditions: Column: Polaris C18-A semi-preparative column. Gradient: 100%-80% A in B from 1-20 min, 80%-50% A in B from 20-40 min, 50%-20% A in B from 40-60 min, and 20%-0% A in B from 60-70 min. Where A = H 2 O (0.1% TFA) and B=CH 3 CN (0.1% TFA); Fig. 9C The HRMS (ESI) spectrum of D-9°NC-3@9-mer is provided: 36 H 59 N 13 O 15 Calculated value of S [M+H] + 946.4053; measured value 946.4064.

[0126] Fig. 10A The analytical HPLC chromatogram (λ=214 nm) of pure D-9°NC-1@33-mer is provided. Column: Welch C4. Gradient: 75%-35% A in B from 0-20 min, 35%-0% A in B from 20-23 min. Where A=H 2 O (0.1% TFA) and B=CH 3CN (0.1% TFA). b) ESI-MS spectrum of D-9°NC-1@33-mer: observed value 3985.5, calculated value 3985.6. Fig. 10B The ESI-MS spectrum of D-9°NC-1@33-mer is provided.

[0127] Fig.11A The analytical HPLC chromatogram (λ=214 nm) of pure D-9°NC-2@39-mer is provided. Column: Welch C4. Gradient: 75%-35% A in B from 0-20 min, 35%-0% A in B from 20-23 min. Where A=H 2 O (0.1% TFA) and B=CH 3 CN (0.1% TFA). Fig. 11B The ESI-MS spectrum of D-9°NC-2@39-mer is provided: observed value 4921.5, calculated value 4921.6.

[0128] Fig. 12A The peptide sequence of D-9°NC-3@21-mer is provided (SEQ ID NO: 15); Fig. 12B An analytical HPLC chromatogram (λ=214 nm) of crude D-9°NC-3@21-mer is provided. Column: Welch C4. Gradient: 90%-50% A in B from 0-30 min, 50%-5% A in B from 30-40 min. Where A=H 2 O (0.1% TFA) and B=CH 3 CN (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 = H 2 O (0.1% TFA) and B=CH 3 CN (0.1% TFA). Fig. 12C The ESI-MS spectrum of D-9°NC-3@21-mer is provided. Fig.12D The MS spectrum of D-9°NC-3@21-mer without TFA protection and the observed mass of 2196.1 are provided.

[0129] Fig.13A is the peptide sequence of D-9°NC-3@Cys35-mer (SEQ ID NO: 16); Fig. 13BAn analytical HPLC chromatogram (λ=214 nm) of crude D-9°NC-3@Cys35-mer is provided. Column: Welch C4. Gradient: 70%-20% A in B from 0-30 min, 20%-5% A in B from 30-40 min. Where A=H 2 O (0.1% TFA) and B=CH 3 CN (0.1% TFA). Purification conditions: Column: Polaris C18-A semi-preparative column. Gradient: 70%-20% A in B from 0-30 min, 20%-5% A in B from 30-40 min. Where A = H 2 O (0.1% TFA) and B=CH 3 CN (0.1% TFA). Fig. 13C The ESI-MS spectrum of D-9°NC-3@21-mer is provided; Fig.13D An observed mass of 4276 was provided for D-9°NC-3@Cys35-mer.

[0130] Fig.14A is the peptide sequence of D-9°NC-3@56-mer (SEQ ID NO: 17); Fig. 14B An analytical HPLC chromatogram (λ=214 nm) of crude D-9°NC-3@56-mer is provided. Column: Welch C4. Gradient: 80%-30% A in B from 0-30 min, 30%-5% A in B from 30-40 min. Where A=H 2 O (0.1% TFA) and B=CH 3 CN (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 = H 2 O (0.1% TFA) and B=CH 3 CN (0.1% TFA).

[0131] Fig.15A is the peptide sequence of D-Pfu-9°NC-3@35-mer (SEQ ID NO: 18); Fig. 15B An analytical HPLC chromatogram (λ=214 nm) of crude D-Pfu-9°NC-3@35-mer is provided. Column: Welch C4. Gradient: 80%-30% A in B from 0-30 min, 30%-5% A in B from 30-40 min. Where A=H 2 O (0.1% TFA) and B=CH 3CN (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 = H 2 O (0.1% TFA) and B=CH 3 CN (0.1% TFA).

[0132] Fig.16A are the peptide sequences of D-9°NN-6@5-mer and D-9°NN-6@11-mer (SEQ ID NO: 19 and SEQ ID NO: 20); Fig. 16B A synthetic route for preparing D-9°NN-6@16-mer is provided. D-9°NN-6@5-mer (4 mg, 1 equivalent) was dissolved in 0.4 mL of acidified ligation buffer (6 M Gn·HCl and 0.1 M NaH 2 PO 4 The mixture was cooled in an ice-salt bath (-15°C) and 40 μl of 0.5 M NaNO 2 The reaction was kept under stirring in an ice-salt bath for 15 min, after which 0.4 ml of 0.2 M MPAA (in 6 M Gn·HCl and 0.1 M Na 2 HPO 4 After adding D-Pfu-9°NN-6@11-mer (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 by TCEP and purified by HPLC (purification conditions: on a Welch C4 column, at 30 min, at 4 °C for 1 h). 2 5%-95% CH in O (with 0.1% TFA) 3 CN (with 0.1% TFA) gradient). The ligation product was obtained in 80% (9.0 mg) yield; Fig. 16C Analytical HPLC chromatograms (λ=214 nm) of peak transitions during the NCL reaction progress are provided. Column: Welch C4. Gradient: at H 2 5%-95% CH 3 CN (with 0.1% TFA) for 30 min.

[0133] Fig.17A are the peptide sequences of D-9°NC-1 and D-9°NC-2 (SEQ ID NOs: 21, 22, 23); Fig. 17BA synthetic route for preparing D-9°NC-7@72-mer is provided. D-9°NC-1@33-mer (5.4 mg) was dissolved in 0.27 mL of acidified ligation buffer (6 M Gn·HCl and 0.1 M NaH 2 PO 4 The mixture was cooled in an ice-salt bath (-15°C) and 27 μl of 0.5 M NaNO 2 (in acidified ligation buffer). The reaction was kept under stirring in an ice-salt bath for 20 min, after which 0.14 ml of 0.4 M MPAA (in 8 M Gn·HCl and 0.1 M Na 2 HPO 4 After adding D-9°NC-2@39-mer (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: on a Polaris C18 column, at H for 30 min. 2 20%-70% CH in O (with 0.1% TFA) 3 CN (with 0.1% TFA) gradient). The ligation product was obtained. Fig. 17C Analytical HPLC chromatogram (λ=214 nm) of the peak transitions during the NCL reaction. Column: Welch C4. Gradient: 2 20%-70% CH 3 CN (with 0.1% TFA) for 30 min. Fig.17D is the ESI-MS of D-9°NC-7@72-mer with MPAA attachment; Fig.17E The MPAA-attached ligation product is shown (observed mass: 9042; calculated mass: 9042).

[0134] 5. Detailed description

[0135] 5.1. Definitions

[0136] "L-nucleic acid" or "L-nucleotide" shall mean any nucleic acid or nucleotide molecule having L chirality, including but not limited to L-DNA, L-RNA and hybrids thereof. The nucleic acid bases forming the nucleic acid molecule may be bases A, C, G, T and U and their derivatives. 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).

[0137] The term "D-polymerase" as used in this application refers to a protein having a mirror image form of an original polymerase having L-chirality. The original polymerase can be a polymerase obtained from nature or modified or synthetically produced. The original polymerase can be a DNA or RNA polymerase.

[0138] Although bases are commonly referred to as purine or pyrimidine bases, it will be appreciated by the skilled artisan that derivatives and analogs that do not change the ability of a nucleotide or nucleoside to undergo Watson-Crick base pairing can be obtained. As used in this application, the term "base" includes compounds or molecules whose core structure is identical or very similar to that of a natural base but which have chemical or physical modifications (such as different or additional side groups that allow a derivative nucleotide or nucleoside to be attached to another molecule). For example, a base can be a deazapurine.

[0139] "Hybridization" shall mean the annealing of a single-stranded nucleic acid to another nucleic acid based on sequence complementarity. The tendency of nucleic acids to hybridize depends on the temperature and ionic strength of their environment, the length of the nucleic acids, and the degree of complementarity. The effects of these parameters on hybridization are 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.).

[0140] Dehybridization is understood by those skilled in the art to mean separation of hybridized primers (or their extensions) from target nucleic acid without destroying the target nucleic acid and thereby allowing further hybridization of a second primer to the target nucleic acid. In one embodiment, hybridization as used in this application means stringent hybridization, such as described in Sambrook, J., Russell, DW, (2000) Molecular Cloning: A Laboratory Manual: 3rd edition.

[0141] As used in this application, hybridization of primer sequences shall mean sufficient annealing such that the primers can be extended by the creation of phosphodiester bonds.

[0142] Where a range of values ​​is provided, it is understood that, unless the context clearly dictates otherwise, each intervening value between the upper and lower limits of the range to the tenth of the unit of the lower limit (if appropriate) and any other stated or intermediate values ​​within the stated range are encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included within these smaller ranges and are also encompassed within the present invention, subject to any specifically excluded limits in the stated ranges. Where the stated range includes one or two limits, ranges excluding one or both of those included limits are also encompassed within the present invention.

[0143] 5.2. Mirror Nucleotides Used in Sequencing

[0144] 5.2.1. L-nucleotides and L-nucleosides

[0145] In one aspect, the present disclosure provides a mirror nucleoside or nucleotide modified to contain at least one of (i) a cleavable protecting group on the 3'O of the pentose ring (if present) and (ii) a cleavable label. The mirror nucleoside or nucleotide can be used in sequencing reactions, polynucleotide synthesis, nucleic acid amplification, nucleic acid hybridization assays, single nucleotide polymorphism studies, techniques using enzymes (such as polymerases, reverse transcriptases, terminal transferases), techniques using labeled dNTPs (e.g., nick translation, random primer labeling, end labeling (e.g., with terminal deoxynucleotidyl transferase)), reverse transcription, or nucleic acid amplification.

[0146] In an embodiment of the invention, a chemically modified mirror nucleoside or nucleotide is provided, which comprises at least one of (i) a cleavable protecting group on the 3'O of the pentose (if present) and (ii) a cleavable label. In embodiments where the mirror nucleotide comprises a cleavable label, the label is not particularly limited, provided that the label provides a means for detection and R' comprises H.

[0147] 5.2.1.1 Bases

[0148] In certain embodiments, the nitrogenous base is a purine or pyrimidine base. In certain embodiments, the nitrogenous base is a deazapurine. In certain embodiments, the nitrogenous base is selected from thymine (5-methyl-2,4-dioxopyrimidine), 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-amino-7-deazapurine), guanine (2-amino-6-oxypurine), 7-deazaguanine (2-amino-4-hydroxy-pyrrolo-[2,3-d]-pyrimidine; 2-amino-7deaza-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.

[0149] 5.2.1.2 R'-cleavable protecting groups

[0150] 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 as long as the resulting nucleotide is an efficient substrate for mirror-image DNA polymerase.

[0151] The skilled artisan will understand how to attach a suitable protecting group to the pentose ring to block the interaction with the 3'-OH. The protecting group may be attached directly at the 3' position or may be attached at the 2' position (the size or charge of the protecting group being sufficient to block the interaction at the 3' position). Alternatively, the protecting group may be attached at both the 3' and 2' positions and may be cleaved to expose the 3' OH group.

[0152] Suitable protecting groups will be apparent to the skilled person and may be formed from any suitable protecting group disclosed in Greene & Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons. The protecting group should be removable (or modifiable) to produce a 3'OH group. The method for obtaining the 3'OH group may be any suitable chemical or enzymatic reaction.

[0153] In some embodiments, the cleavable protecting group comprises an allyl, nitrobenzyl, 1-methyl-2-alkyldisulfide, or methylazido moiety.

[0154] In a specific embodiment, the cleavable protecting group is selected from:

[0155]

[0156] The wavy line marks the point of attachment to 3'O.

[0157] 5.2.1.3 Cleavable Tag Construct (R 2 -mark)

[0158] 5.2.1.3.1 Marking

[0159] Conventional detectable labels may be used with the present invention, which may be modified to be covalently attached to the nucleotide via a cleavable linker.

[0160] Detection can be performed by any suitable method, including fluorescence spectroscopy or by other optical means. Particularly contemplated labels are fluorophores, which 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 that can also be used in the present invention. Labels suitable for use are also disclosed in 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, fluorescein, rhodamine (including TMR, Texas Red and Rox), Alexa Fluor 647, 488, 532, 594, 633; ​​Dyomics - dyes, bodipy, acridine, R6G, Cy3, Cy3.5, Cy5, Cy5.5, coumarin, pyrene, benzanthracene and anthocyanin.

[0161] Although fluorescent labels are specifically contemplated, other forms of detectable labels will be readily apparent to the skilled artisan, 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 labels detectable by changes in pH and voltage, spectroscopic methods such as mass spectrometry, Raman spectroscopy, and surface plasmon resonance (SPR) sensing can all be used.

[0162] i. Fluorophore

[0163] In certain embodiments of the L-nucleotides of the present disclosure, the label is a fluorophore selected from the group consisting of Rox, bodipy (fluoroborate dipyrrole), bodipy-FL-510, R6G, and Cy5, and functional derivatives thereof.

[0164] In specific embodiments, the fluorophore is selected from:

[0165]

[0166] 5.2.1.3.2 Cleavable Linker (R 2 )

[0167] Certain embodiments of L-nucleotides of the present disclosure include a cleavable label connected to, for example, 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 C8 of a purine base, C7 of a 7-deazapurine base, or C5 of a pyrimidine base.

[0168] 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.

[0169] Suitable linkers known to those skilled in the art include, but are not limited to, disulfide linkers, acid-labile linkers (including dialkoxybenzyl linkers, Sieber linkers, indole linkers, tert-butyl Sieber linkers, electrophilic cleavable linkers, nucleophilic cleavable linkers, photocleavable linkers), cleavage under reducing conditions, oxidative conditions, cleavage via the use of safety-catch linkers, and cleavage by an elimination mechanism.

[0170] i. Electrophilic cleavable linker.

[0171] Electrophilic cleavable linkers are typically cleaved by protons and include acid-sensitive cleavage. Suitable linkers include modified benzylic systems such as trityl, p-alkoxybenzyl esters and p-alkoxybenzyl amides. Other suitable linkers include tert-butoxycarbonyl (Boc) and acetal systems.

[0172] It is also contemplated to use thiophilic metals such as nickel, silver or mercury in the cleavage of thioacetals or other sulfur-containing protecting groups for the preparation of suitable linker molecules.

[0173] ii. Nucleophilic cleavage of the linker.

[0174] Nucleophilic cleavage is also a recognized method in the preparation of linker molecules. Groups such as unstable esters in water (i.e., can be simply cleaved at alkaline pH) and groups unstable to non-aqueous nucleophiles can be used. Fluoride ions can be used to cleave silicon-oxygen bonds in groups such as triisopropylsilane (TIPS) or tert-butyldimethylsilane (TBDMS).

[0175] iii. Photocleavable linkers.

[0176] Photocleavable linkers have been widely used in carbohydrate chemistry. Preferably, the light required for cleavage activation does not affect other components of the modified nucleotide. For example, if a fluorophore is used as a label, it is preferred if the wavelength of light absorbed by it is different from the wavelength required for the cleavage linker molecule. Suitable linkers include those based on O-nitrobenzyl compounds and nitroveratrol compounds. Linkers based on benzoin chemistry can also be used (Lee et al., J. Org. Chem. 64: 3454–3460, 1999).

[0177] iv. Cleavage under reducing conditions

[0178] Many linkers are known to be susceptible to reductive cleavage. Catalytic hydrogenation using palladium-based catalysts has been used to cleave benzyl and benzyloxycarbonyl groups. Disulfide bond reduction is also known in the art.

[0179] v. Cleavage under oxidative conditions

[0180] Oxidation-based methods are well known in the art. These include oxidation of alkoxybenzyl groups and oxidation of sulfur and selenium linkers. The use of aqueous iodine to cleave disulfide groups and other sulfur or selenium-based linkers is also within the scope of the present invention.

[0181] vi. Safety-catch connector

[0182] Safe capture linkers are those that cleave in two steps. In a preferred system, the first step is to create a reactive nucleophilic center, followed by a second step involving intramolecular cyclization that leads to cleavage. For example, the levulinate bond can be treated with hydrazine or photochemically to release the active amine, which is then cyclized to cleave the ester elsewhere in the molecule (Burgess et al., J. Org. Chem. 62: 5165–5168, 1997).

[0183] vii. Cleavage by Elimination Mechanism

[0184] Elimination reactions may also be used. For example, base catalyzed elimination of groups such as Fmoc and cyanoethyl, and palladium catalyzed reductive elimination of allylic systems may be used.

[0185] 5.2.1.3.3 Spacer

[0186] In addition to the cleavage site, the linker may include one or more spacer moieties. The spacer (linker and bridge) may be any typically used in, for example, the synthesis of a bioconjugate. The spacer keeps the nucleotide bases away from the cleavage site or label. The length of the linker is not important, provided that the label is kept at a sufficient distance from the nucleotides so as not to interfere with any interaction between the nucleotides and the enzyme.

[0187] In specific embodiments, the cleavable linker comprises, in addition to an additional spacer, an allyl group, a disulfide group, or an azide group. In certain embodiments, the cleavable linker comprises:

[0188]

[0189] 5.2.1.3.4 Cleavable Tag Constructs

[0190] In certain embodiments of the mirror nucleosides or nucleotides provided herein, the cleavable label comprises a label as described in Section 5.2.1.3.1 and a cleavable linker as described in more detail in Section 5.2.1.3.2.

[0191] In specific embodiments, the cleavable tag construct is selected from:

[0192]

[0193] Wherein the wavy line indicates the point of attachment to the mirror nucleotide, including attachment to a linker / spacer that is attached to the nucleotide for the purpose of enabling attachment, e.g., an amine moiety.

[0194] 5.2.2. Implementation of the present disclosure

[0195] In certain embodiments, an L-nucleoside or L-nucleotide is provided, comprising:

[0196] A pentose selected from the group consisting of (3R,4S)-3,4,5-trihydroxypentanal and (4R)-4,5-dihydroxypentanal;

[0197] wherein C-5 of the sugar is substituted with a hydroxyl group, a monophosphate, a diphosphate, or a triphosphate; and

[0198] The C-3 of the sugar is substituted with H or O-R', wherein R' is H or a cleavable protecting group;

[0199] Nitrogenous bases; and

[0200] an optional cleavable label;

[0201] The L-nucleoside or L-nucleotide comprises at least one selected from (i) a cleavable protecting group and (ii) a cleavable label.

[0202] The embodiments provided in this application will be further described with reference to nucleotides. However, unless otherwise specified, references to nucleotides are also intended to apply to nucleosides.

[0203] In certain embodiments, an L-nucleotide having a structure according to Formula A is provided:

[0204]

[0205] wherein R' is H or a cleavable protecting group; X is H or hydroxyl, BASE is a nitrogenous base, and wherein the nucleotide optionally comprises a cleavable label, and wherein at least one of a cleavable protecting group or a cleavable label is present.

[0206] In certain embodiments, the nitrogenous base is as described in Section 5.2.1.1 of this application. In certain embodiments, the cleavable protecting group is as described in Section 5.2.1.2 of this application. In certain embodiments, the cleavable label is as described in Section 5.2.1.3 of this application.

[0207] In any of the described embodiments, the position of the cleavable label is not particularly limited and can be attached to the nucleotide at any position that is chemically feasible and / or known in the art. In certain embodiments, the cleavable label is attached to the nitrogenous base through a terminal phosphate or at the 3'O position, in which case the cleavable protecting group is a cleavable label.

[0208] 5.2.2.1 (L)-Nucleotide reversible terminator (L-NRT)

[0209] 5.2.2.1.1 [(L)-3'-O-R'-dNTPs] - Formula I

[0210] In one aspect of the present disclosure, there is provided an L-nucleotide having a structure according to Formula I:

[0211]

[0212] wherein the base is a nitrogenous base and R' is a cleavable protecting group.

[0213] In certain embodiments, the nitrogenous base is as described in Section 5.2.1.1 of this application. In certain embodiments, the cleavable protecting group is as described in Section 5.2.1.2 of this application.

[0214] In certain embodiments, L-nucleotides are provided having a structure according to:

[0215]

[0216] In certain embodiments, R' comprises a cleavable protecting group as described in Section 5.2.1.2 of this application. In other embodiments, the cleavable protecting group R' is selected from:

[0217]

[0218] The wavy line marks the point of attachment to 3'O.

[0219] 5.2.2.1.2[(L)-3'-O-R'-dNTP-R 2 -label]-Formula II

[0220] In one aspect of the present disclosure, there is provided an L-nucleotide having a structure according to Formula II:

[0221]

[0222] wherein the base is a nitrogenous base; R' is a cleavable protecting group or H; and the label -R 2 A cleavable tag construct is included together.

[0223] In certain embodiments, the nitrogenous base is as described in Section 5.2.1.1 of this application. In certain embodiments, the cleavable protecting group is as described in Section 5.2.1.2 of this application. In certain embodiments, the cleavable tag construct is as described in Section 5.2.1.3 of this application.

[0224] In certain embodiments, an L-nucleotide having a structure selected from the group consisting of:

[0225]

[0226] In certain embodiments, the cleavable protecting group R' is as described in Section 5.2.1.2 of this application. In certain embodiments, the cleavable linker R 2 As described in Section 5.2.1.3.2 of this application.

[0227] In specific embodiments, R' is selected from:

[0228] H.

[0229] The wavy line marks the point of attachment to 3'O.

[0230] In a specific embodiment, R 2 Include:

[0231]

[0232] In a specific embodiment, an L-nucleotide having a structure selected from the group consisting of:

[0233]

[0234]

[0235] wherein R' is H or a cleavable protecting group as described in Section 5.2.1.2 of this application.

[0236] In certain embodiments, R' is selected from:

[0237]

[0238] The wavy line marks the point of attachment to 3'O.

[0239] 5.2.2.2 Marked terminator

[0240] 5.2.2.2.1[(L)-ddNTP-R 2 -labeled] Formula III

[0241] In one aspect of the present disclosure, there is provided an L-nucleotide having a structure according to Formula III:

[0242]

[0243] The base is a nitrogenous base; R 2 is a cleavable linker; and labeled -R 2 A cleavable tag construct is included together.

[0244] In certain embodiments, the nitrogenous base is as described in Section 5.2.1.1 of this application. In certain embodiments, the cleavable tag construct is Tag-R 2As described in Section 5.2.1.3 of this application.

[0245] In certain embodiments, an L-nucleotide having a structure selected from the group consisting of:

[0246]

[0247] In certain embodiments, the cleavable linker R 2 As described in Section 5.2.1.3.2 of this application.

[0248] In a specific embodiment, R 2 Include:

[0249]

[0250] In a specific embodiment, an L-nucleotide having a structure selected from the group consisting of:

[0251]

[0252]

[0253] Mirror Image Polymerase

[0254] In another aspect, the present disclosure provides a mirror image polymerase. Specifically, the mirror image polymerase comprises D-type amino acids. In some embodiments, the mirror image polymerase consists of D-type amino acids. In some embodiments, the mirror image polymerase comprises both D-type and L-type amino acids. In some embodiments, the mirror image polymerase does not comprise L-type amino acids.

[0255] In some embodiments, the mirror nucleic acid is a mirror image of a DNA polymerase or RNA polymerase. In some embodiments, the mirror nucleic acid is a mirror image of a 9°N DNA polymerase or a modification thereof.

[0256] In some embodiments, the mirror image polymerase comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror image polymerase comprises a sequence having at least 96% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror image polymerase comprises a sequence having at least 96% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror image polymerase comprises a sequence having at least 97% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror image polymerase comprises a sequence having at least 98% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror image polymerase comprises a sequence having at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror image polymerase comprises a sequence of SEQ ID NO: 1.

[0257] In some embodiments, the mirror image polymerase has a sequence with at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror image polymerase has a sequence with at least 96% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror image polymerase has a sequence with at least 96% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror image polymerase has a sequence with at least 97% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror image polymerase has a sequence with at least 98% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror image polymerase has a sequence with at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror image polymerase has a sequence of SEQ ID NO: 1.

[0258] 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.

[0259]

[0260] In some embodiments, the mirror image polymerase comprises one or more modifications for native chemical ligation (NCL) compared to SEQ ID NO: 1. In some embodiments, the mirror image polymerase comprises one or more modifications at one or more amino acid positions selected from E276, K317, N424, and S651 compared to SEQ ID NO: 1. In some embodiments, the mirror image polymerase comprises one or more substitutions selected from E276A, K317G, N424A, and S651A compared to SEQ ID NO: 1. In some embodiments, the mirror image polymerase comprises E276A, K317G, N424A, and S651A substitutions compared to SEQ ID NO: 1.

[0261] In some embodiments, the mirror image polymerase comprises one or more substitutions of isoleucine compared to SEQ ID NO: 1. In some embodiments, the mirror image polymerase comprises one or more modifications at one or more amino acid positions 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 from Ile to Ala, Val, Leu, or Tyr at one or more amino acid positions selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733, and I744, as 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.

[0262] In some embodiments, the mirror image polymerase comprises one or more modifications compared to SEQ ID NO: 1 to improve its interaction with the L-nucleotides or modifications thereof disclosed in this application.

[0263] In some embodiments, the mirror image polymerase comprises one or more modifications at one or more amino acid positions 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 image polymerase comprises one or more modifications at one or more amino acid positions selected from D141, E143, Y409, and A485 compared to SEQ ID NO: 1. In some embodiments, the mirror image polymerase comprises one or more substitutions selected from D141A, E143A, Y409V, and A485L compared to SEQ ID NO: 1. In some embodiments, the mirror image polymerase comprises substitutions of D141A, E143A, Y409V, and A485L compared to SEQ ID NO: 1.

[0264] In some embodiments, the mirror image polymerase comprises one or more modifications at one or more amino acid positions selected from D141, E143, L408, Y409, P410, A485, T514, and I521 compared to SEQ ID NO: 1. In some embodiments, the mirror image 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 image polymerase comprises substitutions of D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L compared to SEQ ID NO: 1.

[0265] In some embodiments, the mirror image polymerase comprises one or more modifications selected from the group consisting of substitutions of M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S, I521L, or an addition of 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 an addition of D between I130 and G131 compared to SEQ ID NO: 1.

[0266] 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.

[0267] Mirror image polymerase can be obtained by chemical synthesis. In some embodiments, polypeptides or small peptides are synthesized by solid phase peptide synthesis and then connected by chemical ligation to obtain mirror image polymerase. In some embodiments, the mirror image polymerase has the same enzymatic activity as the original polymerase, but acts on D-type nucleotides.

[0268] 5.4. Usage

[0269] In one aspect, the present disclosure provides methods of using the mirror nucleotides and mirror polymerases disclosed in the present application. The mirror compositions can be used to replicate or sequence L-polynucleotides. In some embodiments, the compositions are used in sequencing methods adapted from sequencing by synthesis methods, Sanger methods, or a combination thereof.

[0270] 5.4.1. Method of copying L-shaped template

[0271] The present disclosure provides a method for replicating an L-polynucleotide. The method may include the following steps: incubating a mixture comprising (i) an L-polynucleotide, (ii) an L-primer, (iii) an L-dNTP, (iv) a mirror-image polymerase, and (v) a buffer, thereby inducing replication of the L-polymerase. In some embodiments, the L-polymerase is an L-type DNA or RNA. In some embodiments, the L-polymerase comprises at least 10, 20, 30, 50, 100, 200, 500 or more nucleotides.

[0272] 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.

[0273] In some embodiments, the mixture comprises a buffer developed for use with 9 ° N DNA polymerase. In some embodiments, the buffer comprises 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 comprises 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 comprises MgCl 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. 2 In some embodiments, the buffer comprises MgCl at a concentration of about 10 mM, 20 mM, 30 mM, 40 mM, or 50 mM. 2 In some embodiments, the buffer comprises MnCl 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. 2 In some embodiments, the buffer comprises MnCl at a concentration of about 10 mM, 20 mM, 30 mM, 40 mM, or 50 mM. 2. In some embodiments, the buffer comprises DTT at a concentration of 0.1mM to 10mM, 0.5mM to 5mM, 0.5mM to 3mM, or 0.5mM to 2mM. In some embodiments, the buffer comprises DTT at a concentration of about 0.5mM, 1mM, 1.5mM, or 2mM. In some embodiments, the buffer comprises KCl at a concentration of 10mM to 100mM, 20mM to 80mM, 25mM to 75mM, or 30mM to 70mM. In some embodiments, the buffer comprises KCl at a concentration of about 25mM, 30mM, 40mM, 50mM, 60mM, 70mM, or 100mM. 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.

[0274] In some embodiments, the buffer comprises Tris-HCl, MgCl 2 , DTT and KCl. In some embodiments, the buffer comprises Tris-HCl, MnCl 2 , DTT and KCl. In some embodiments, the buffer comprises Tris-HCl, MnCl 2 MgCl 2 , DTT and KCl. In some embodiments, the buffer comprises 50 mM Tris-HCl, pH 7.5, 20 mM MgCl 2 , 1 mM DTT and 50 mM KCl.

[0275] Sequencing methods

[0276] The present disclosure provides a method for sequencing an L-polynucleotide. The L-polynucleotide comprises an L-type nucleotide. The L-polynucleotide can be DNA or RNA.

[0277] In some embodiments, the sequencing method comprises the following cycles: (a) adding a sequence comprising (i) an L-polynucleotide, (ii) an L-primer, (iii) an L-3'-O-R'-dNTP-R 2 -label, (iv) a mirror-image polymerase and (v) a buffer, thereby obtaining a replication product; (b) detecting L-3'-O-R'-dNTP-R incorporated into the replication product 2 -labeled signal; and (iii) inducing incorporation of L-3'-O-R'-dNTP-R into the replication product 2 - labeled R' groups and R 2The method comprises (i) incorporating an L-3'-O-R'-dNTP-R2-label, (ii) identifying the incorporated nucleotide by a signal from the incorporated L-3'-O-R'-dNTP-R2-label, and (iii) cleaving the label while restarting the polymerase reaction for continuous sequence determination. The L-3'-O-R'-dNTP-R2-label comprises a chemical moiety (R') that caps the 3'-OH and a chemically cleavable linker (R 2 ) is tethered to the label of the base. The 3'-capping portion (R') and the label on the reaction product are cleaved to allow the polymerase reaction to restart.

[0278] In some embodiments, the method comprises 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.

[0279] 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.

[0280] In some embodiments, L-3'-O-R'-dNTP-R 2 - Label contains L-3'-O-R'-dATP-R 2 -label, L-3'-O-R'-dTTP-R 2 -label, L-3'-O-R'-dGTP-R 2 -Tag or L-3'-O-R'-dCTP-R 2 -label. In some embodiments, L-3'-O-R'-dNTP-R 2 - Label contains L-3'-O-R'-dATP-R 2 -label, L-3'-O-R'-dTTP-R 2 -label, L-3'-O-R'-dGTP-R 2 -Tag and L-3'-O-R'-dCTP-R 2 - two or more of the labels. In some embodiments, L-3'-O-R'-dNTP-R 2- Label contains L-3'-O-R'-dATP-R 2 -label, L-3'-O-R'-dTTP-R 2 -label, L-3'-O-R'-dGTP-R 2 -Tag and L-3'-O-R'-dCTP-R 2 - Label. The label attached to each dNTP can be unique and identifiable.

[0281] In some embodiments, L-3'-O-R'-dNTP-R 2 - The label has a structure according to Formula I as described in 5.2.2.1.2. In some embodiments, L-3'-O-R'-dNTP-R 2 - The tag has a structure according to Formula II as described in 5.2.2.1.2.

[0282] In some embodiments, L-3'-O-R'-dNTP-R 2 - The 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-R 2 -Labels (e.g., dATP, dGTP, dTTP, dCTP) are labeled as unique labels. In some embodiments, each type of L-3'-O-R'-dNTP-R 2 -Label (e.g., dATP, dGTP, dTTP, dCTP) provides a unique fluorescent signal. L-3'-O-R'-dNTP-R 2 - The label may include any label disclosed in 5.2.1.3.1. In some embodiments, L-3'-O-R'-dNTP-R 2 -Labels include non-fluorescent labels.

[0283] In some embodiments, the sequencing method comprises the following steps: (a) adding a sequence comprising (i) L-polynucleotide, (ii) L-primer, (iii) L-dNTP, (iv) L-ddNTP-R 2 -label, (v) the mirror polymerase disclosed in the present application and (vi) a buffer, thereby obtaining a replication product; (b) isolating the replication product; and (c) detecting the L-ddNTP-R incorporated into the replication product. 2 - The labeled signal. The incubation step may comprise a PCR reaction.

[0284] In a preferred embodiment of the method, a low ratio of L-ddNTP-R is added to the mixture compared to L-dNTP. 2-labeling. During incubation (e.g., PCR reaction), L-ddNTP-R lacking a 3'-OH group 2 - The label can be randomly incorporated into the replication product by the mirror image polymerase. L-ddNTP-R 2 -label incorporation terminates the replication process. The reaction can induce the production of oligonucleotide copies of the replication product, which are randomly ligated by L-ddNTP-R 2 - Marks the end.

[0285] In this method, the replication products can be separated by size. In some embodiments, the replication products are separated by size via gel electrophoresis. 2 - Labeled signal that can determine the end of the replication product L-ddNTP-R 2 -The identity of the label (e.g., ddATP, ddGTP, ddTTP or ddCTP). The data can show the nucleotide types along the length of the L-polynucleotide. This process can allow the sequence of the L-polynucleotide to be determined.

[0286] 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.

[0287] In some embodiments, L-ddNTP-R 2 - Tag contains L-ddATP-R 2 -Marker, L-ddTTP-R 2 -label, L-ddGTP-R 2 -Mark or L-ddCTP-R 2 -label. In some embodiments, L-ddNTP-R 2 - Tag contains L-ddATP-R 2 -Marker, L-ddTTP-R 2 -label, L-ddGTP-R 2 -Marker and L-ddCTP-R 2 - one or more of the labels. In some embodiments, L-ddNTP-R 2 - Tag contains L-ddATP-R 2 -Marker, L-ddTTP-R 2 -label, L-ddGTP-R 2 -Marker and L-ddCTP-R2 - Label. The label attached to each ddNTP can be unique and identifiable.

[0288] In some embodiments, L-ddNTP-R 2 - The tag has the structure disclosed in 5.2.2.2.1. In some embodiments, L-ddNTP-R 2 - The marker has the formula III as defined in 5.2.2.2.1.

[0289] In some embodiments, L-ddNTP-R 2 - The label comprises a fluorescent label. In this case, the signal is a fluorescent signal. In some embodiments, each type of L-ddNTP-R 2 -Labels (e.g., ddATP, ddGTP, ddTTP, ddCTP) are labeled as unique labels. In some embodiments, each type of L-ddNTP-R 2 -Label (e.g., ddATP, ddGTP, ddTTP, ddCTP) provides a unique fluorescent signal. L-ddNTP-R 2 - The label may include any label disclosed in 5.2.1.3.1. In some embodiments, L-ddNTP-R 2 -Labels include non-fluorescent labels.

[0290] In some embodiments, the sequencing method comprises the following cycles: (a) adding a sequence comprising (i) L-polynucleotide, (ii) L-primer, (iii) L-3'-O-R'-dNTP, (iv) L-ddNTPs-R 2 -label, (v) the mirror polymerase provided in the present application and (vi) a buffer mixture incubated to obtain a replication product; (b) detecting the L-ddNTP-R incorporated into the replication product 2 - labeled signal; and (c) inducing the incorporation of the R' group of the L-3'-O-R'-dNTP and the L-ddNTPs-R 2 -Marked R 2 In this method, the polymerase reaction is activated by a 3′-capped nucleotide reversible terminator (L-3′-O-R′-dNTP) and a cleavable fluorescent dideoxynucleotide (L-ddNTPs-R 2 -labeled) combination. In this method, the sequence is composed of ddNTPs (L-ddNTPs-R 2 The signal of each label on the reaction product terminated by the incorporation of a 3′-OH capping group (R′ group) from the incorporated nucleotide reversible terminator (L-3′-O-R′-dNTP) and the signal of each label on the reaction product terminated by the incorporation of a 3′-OH capping group (R′ group) from the incorporation of a 3′-OH capping group ...2 After the tag is removed from the terminated DNA product, the polymerase reaction is restarted to continue sequence determination.

[0291] In some embodiments, the method comprises 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.

[0292] 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.

[0293] In some embodiments, 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, 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, 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.

[0294] In some embodiments, L-3'-O-R'-dNTP has any of the structures provided in 5.2.2.1.1. In some embodiments, L-3'-O-R'-dNTP has Formula I as defined in 5.2.2.1.1.

[0295] In some embodiments, L-ddNTP-R 2 - Tag contains L-ddATP-R 2 -Marker, L-ddTTP-R 2 -label, L-ddGTP-R 2 -Mark or L-ddCTP-R 2 -label. In some embodiments, L-ddNTP-R 2- Tag contains L-ddATP-R 2 -Marker, L-ddTTP-R 2 -label, L-ddGTP-R 2 -Marker and L-ddCTP-R 2 - one or more of the labels. In some embodiments, L-ddNTP-R 2 - Tag contains L-ddATP-R 2 -Marker, L-ddTTP-R 2 -label, L-ddGTP-R 2 -Marker and L-ddCTP-R 2 - Label. The label attached to each dNTP can be unique and identifiable.

[0296] In some embodiments, L-ddNTP-R 2 - The tag has any of the structures provided in 5.2.2.2.1. In some embodiments, L-ddNTP-R 2 - The label has Formula III as defined in 5.2.2.2.1. In some embodiments, each type of L-3'-O-R'-dNTP-R 2 -Label (e.g., dATP, dGTP, dTTP, dCTP) provides a unique fluorescent signal. L-ddNTP-R 2 - The label may include any label disclosed in 5.2.1.3.1. In some embodiments, L-ddNTP-R 2 -Labels include non-fluorescent labels.

[0297] In some embodiments, the sequencing method comprises the following cycles: (a) incubating a mixture comprising (i) an L-polynucleotide, (ii) an L-primer, (iii) an L-dNTP, (iv) a mirror-image polymerase, and (v) a buffer to obtain a reaction product; and (b) detecting the release of PPi molecules in the reaction product. In some embodiments, the sequencing method comprises multiple cycles, wherein 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, wherein each cycle is performed by stepwise changing the type of L-dNTP to one of L-dATP, L-dTTP, L-dGTP, and L-dCTP.

[0298] In some embodiments, the step of detecting the release of the PPi molecule comprises the following steps: (i) treating the reaction product to convert the PPi molecule ATP (if present) and generating a detectable signal by the ATP (if present); and (ii) detecting the presence or absence of the detectable signal. In some embodiments, ATP is treated with 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.

[0299] 5.5. Kit

[0300] The present disclosure provides a kit for replicating or sequencing an L-polymerase. The kit comprises the mirror image polymerase disclosed in the present application and an optional buffer.

[0301] In some embodiments, the kit further comprises L-dNTP. In some embodiments, L-NTP comprises L-dATP, L-dGTP, L-dCTP, L-dTTP or L-UTP. In some embodiments, L-NTP comprises one or more of L-dATP, L-dGTP, L-dCTP, L-dTTP and L-UTP. In some embodiments, L-NTP comprises L-dATP, L-dGTP, L-dCTP and L-dTTP. In some embodiments, L-NTP comprises L-dATP, L-dGTP, L-dCTP and L-UTP. In some embodiments, L-NTP comprises L-dATP, L-dGTP, L-dCTP, L-dTTP and L-UTP.

[0302] In some embodiments, the kit comprises L-3'-O-R'-dNTP-R 2 -label. In some embodiments, L-3'-O-R'-dNTP-R 2 - Label contains 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 -label. In some embodiments, L-3'-O-R'-dNTP-R 2 - Label contains 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 -labeled and L-3'-O-R'-dUTP-R 2 - one or more of the labels. In some embodiments, L-3'-O-R'-dNTP-R 2 - Label contains L-3'-O-R'-dATP-R 2 -label, L-3'-O-R'-dTTP-R 2 -label, L-3'-O-R'-dGTP-R 2 -Tag and L-3'-O-R'-dCTP-R 2 - one or more of the labels. In some embodiments, L-3'-O-R'-dNTP-R 2 - Label contains L-3'-O-R'-dATP-R 2 -label, L-3'-O-R'-dGTP-R 2 -label, L-3'-O-R'-dCTP-R 2 -labeled and L-3'-O-R'-dUTP-R 2 - one or more of the labels. In some embodiments, each type of L-3'-O-R'-dNTP-R 2 - The label (e.g., dATP, dGTP, dTTP, dCTP) provides a unique fluorescent signal.

[0303] In some embodiments, L-3'-O-R'-dNTP-R 2 - The label has Formula II as defined in 5.2.2.1.2. In some embodiments, L-3'-O-R'-dNTP-R 2 - The tag has any structure disclosed in 5.2.2.1.2.

[0304] In some embodiments, the kit comprises L-ddNTP-R 2 -label. In some embodiments, L-ddNTP-R 2 - Tag contains L-ddATP-R 2 -Marker, L-ddTTP-R 2 -label, L-ddGTP-R 2 -Marker, L-ddCTP-R 2 - Marker or L-ddUTP-R 2 - Label. L-ddNTP-R 2 -label. In some embodiments, L-ddNTP-R 2 - Tag contains L-ddATP-R 2 -Marker, L-ddTTP-R2 -label, L-ddGTP-R 2 -Marker, L-ddCTP-R 2 -Marker and L-ddUTP-R 2 - one or more of the labels. In some embodiments, L-ddNTP-R 2 - Tag contains L-ddATP-R 2 -Marker, L-ddTTP-R 2 -label, L-ddGTP-R 2 -Marker and L-ddCTP-R 2 -label. In some embodiments, L-ddNTP-R 2 - Tag contains L-ddATP-R 2 -label, L-ddGTP-R 2 -Marker, L-ddCTP-R 2 -Marker and L-ddUTP-R 2 -mark.

[0305] In some embodiments, L-ddNTP-R 2 - The label has Formula III as defined in 5.2.2.2.1. In some embodiments, L-ddNTP-R 2 - The tag has any structure disclosed in 5.2.2.2.1.

[0306] In some embodiments, the kit further comprises L-3'-O-R'-dNTP. L-3'-O-R'-dNTP may comprise L-3'-O-R'-dATP, L-3'-O-R'-dTTP, L-3'-O-R'-dGTP or L-3'-O-R'-dCTP. In some embodiments, 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, 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.

[0307] In some embodiments, L-3'-O-R'-dNTP has Formula I as defined in 5.2.2.1.1. In some embodiments, L-3'-O-R'-dNTP has any of the structures disclosed in 5.2.2.1.1.

[0308] In some embodiments, every kind of L-nucleotide-label in the test kit has a unique label. In some embodiments, some of the L-nucleotide-label in the test kit have the same label. In some embodiments, the label is a fluorescent label. In some embodiments, every kind of L-nucleotide-label in the test kit provides a unique fluorescent signal. 6. Examples

[0309] 6.1. Example 1. Synthesis of (L)-3'-O-azidomethyl-dNTP

[0310] Materials and methods

[0311] Starting materials β-L-deoxyadenosine, L-deoxyguanosine, L-deoxycytidine, L-deoxythymidine can be purchased from, for example, Chemgenes. Unless otherwise stated, all solvents and reagents were of reagent grade, purchased commercially, and used without further purification.

[0312] 6.1.1. (L) Synthesis of 3'-O-azidomethyl-dATP

[0313] 6.1.1.1 Scheme 1. (L)3'-ON 3 -dATP synthesis

[0314]

[0315] A stirred solution of 'starting material' (β-L-deoxyadenosine) (1.0 eq., 2.00 g, 7.48 mmol) was co-evaporated with anhydrous pyridine, dissolved in 15 ml of anhydrous pyridine and sealed with a septum. After stirring for 5 minutes under argon, trimethylsilyl chloride (TMS-Cl, 5.0 eq., 4.06 g, 37.4 mmol) was added via syringe. Benzoyl chloride (1.2 eq., 1.26 g, 8.98 mmol) was added dropwise via syringe over a period of 20 minutes and after 30 minutes, and stirring was continued for 2.5 h while a clear yellow solution was formed. Then, 4 ml of H was added at once. 2 O, and after 5 minutes, 8 ml of aqueous ammonia solution (28%-30%) was added at a time and stirred for another 15 minutes. The mixture was evaporated to dryness, and the oily residue was co-evaporated twice with toluene to obtain 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) was added tert-butyldimethylsilyl chloride (TBDMSCl) (765 mg; 4.92 mmol). The reaction mixture was stirred at room temperature for 20 h. After evaporation, the residue was purified by flash column chromatography using CH 3OH-CH 2 Cl 2 Purification was performed with 4% paraformaldehyde (1:20) as the eluent to obtain 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) were added acetic acid (5.5 ml) and acetic anhydride (17.6 ml). The reaction mixture was stirred at room temperature for 48 h. Saturated NaHCO 3 solution (100 ml) and the aqueous layer was washed with CH 2 Cl 2 The combined organic extracts were washed with saturated NaHCO 3 solution (100 ml) and washed with Na 2 SO 4 After concentration, the residue was purified by flash column chromatography (hexane / ethyl acetate, 1:1 to 1:4) to give compound dA-3 in the form of a white powder. Under nitrogen, compound dA-3 (400 mg; 0.76 mmol) was added to dry CH 2 Cl 2 To a stirred solution of 1% cyclohexene (400 μl) and SO 2 Cl 2 (155 μl; 1.91 mmol, redistilled). The reaction mixture was stirred at 0°C for 2 h. The solvent was removed first under reduced pressure and then under a high vacuum pump for 10 min. The residue was dissolved in dry DMF (5 ml) and stirred at room temperature with NaN 3 (400 mg; 6.6 mmol) was reacted for 3 h. The reaction mixture was dispersed in distilled water (50 ml) and washed with CH 2 Cl 2 (3×50 ml) extraction. The combined organic layers were purified by Na 2 SO 4 The residue was dissolved in MeOH (5 ml) and stirred at room temperature with NH 4 F (300 mg; 8.1 mmol) was stirred for 24 h. The solvent was removed under reduced pressure. The reaction mixture was concentrated under reduced pressure and 2 O and CH 2 Cl 2 The organic layer was separated and purified by Na 2 SO 4 After concentration, the crude product was purified by flash column chromatography (ethyl acetate / methanol, 100:0 to 98:2) to obtain compound dA-4 in the form of a white powder. Compound dA-4 (123 mg; 0.3 mmol) and proton sponge (75.8 mg; 0.35 mmol) were mixed in a vacuum desiccator over P2 O 5 Dry overnight and then dissolve in trimethyl phosphate (600 ml). Freshly distilled POCl was then added dropwise at 0 °C. 3 (40 μl; 0.35 mmol) and the mixture was stirred at 0°C for 2 h. Subsequently, a well-vortexed mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 ml; 2.31 mmol) in anhydrous DMF (2.33 ml) was added all at once and stirred for 30 min at room temperature. Triethylammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 ml) was then added and the mixture was stirred at room temperature for 1 h. Conc. NH 4 OH (15 ml) and stirred at room temperature overnight. The resulting mixture was concentrated under vacuum and the residue was diluted with 5 ml of water. The crude mixture was 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 was further purified by reverse phase HPLC to give (L) 3'-O-azidomethyl-dATP (compound dA-5).

[0316] 6.1.2. (L) Synthesis of 3'-O-azidomethyl-dGTP

[0317] 6.1.2.1 Scheme 2. (L)3'-ON 3 Synthesis of -dGTP

[0318]

[0319] A stirred solution of 'starting material' (β-L-deoxyguanosine) (1.0 eq., 2.00 g, 7.13 mmol) was co-evaporated with anhydrous pyridine (3×4 mL) and then dissolved in anhydrous pyridine (2 mL). The resulting solution was 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 eq.) was added dropwise via a syringe. The ice bath was then removed and the mixture was stirred for 2 hours. The solution was cooled on ice, and isobutyric anhydride (0.29 mL, 15.69 mmol, 2.2 eq.) was added dropwise via a syringe and the ice bath was removed. After stirring for another 2 hours at room temperature, the reaction was again placed on ice, and ice-cold water (20 mL) was slowly added, followed by concentrated ammonia solution (1.5 mL) after 15 minutes to give a final 2.5 M concentration of ammonia. The mixture was kept on ice for 30 minutes and then evaporated to dryness. The residue was co-evaporated with toluene (3×5 mL) to remove traces of water, resuspended in MeOH and filtered to remove precipitate. The filtrate was then concentrated, dissolved in a small amount of MeOH, adsorbed on silica gel and purified by column chromatography (DCM / MeOH 95:5 to 91:9 (v / v)) to give compound dG-1 in the form of a yellow solid. Compound dG-1 (495 mg, 1.07 mmol) was co-evaporated three times with dry pyridine, dried under high vacuum, and dissolved in 2 cm 4% 4% 4% pyridine in an ice bath. 3 In dry N, N-dimethylformamide. Di-tert-butylsilyl bis(trifluoromethanesulfonic acid) ester (590mg, 1.34mmol) was added dropwise over a period of 15min, and the reaction mixture was stirred at 0°C for 30min. Imidazole (419mg, 6.15mmol) was added, and the mixture was stirred at 0°C for 15min and at room temperature for 15min. Then, 241mg tert-butyldimethylsilyl chloride (1.59mmol) was added, and the solution was stirred for another 2h at 60°C. The mixture was diluted with dichloromethane, washed with brine, dried over sodium sulfate, and evaporated. The crude product was purified as a white foam (compound dG-2) by column chromatography on silica gel (methanol: dichloromethane 0:100–2:98). Acetic acid (5.5ml) and acetic anhydride (17.6ml) were added to a stirred solution of compound dG-2 (3.0g; 6.08mmol) in DMSO (12ml). The reaction mixture was stirred at room temperature for 48 h. Saturated NaHCO 3 solution (100 ml) and the aqueous layer was washed with CH 2 Cl 2 The combined organic extracts were washed with saturated NaHCO 3 solution (100 ml) and washed with Na 2 SO4 Dry. After concentration, the residue was purified by flash column chromatography (hexane / ethyl acetate, 1: 1 to 1: 4) to give compound dG-3 in the form of a white powder. To a stirred solution of compound dG-3 (1.0 g; 2.0 mmol) in dry pyridine (22 ml) were added diphenylcarbamoyl chloride (677 mg; 2.92 mmol) and DIEA (N,N-diisopropylethylamine) (1.02 ml; 5.9 mmol). The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 3 h. The solvent was removed under high vacuum. The crude product was purified by flash column chromatography (ethyl acetate / hexane, 1: 1 to 7: 3) to give compound dG-4 in the form of a light yellow powder. Under nitrogen, compound dG-4 (400 mg; 0.71 mmol) was stirred in dry CH 2 Cl 2 To a stirred solution of 1% cyclohexene (400 μl) and SO 2 Cl 2 (155 μl; 1.91 mmol, redistilled). The reaction mixture was stirred at 0°C for 2 h. The solvent was removed first under reduced pressure and then under high vacuum pump for 10 min. The residue was dissolved in dry DMF (5 ml) and mixed with NaN at room temperature. 3 (400 mg; 6.6 mmol) was reacted for 3 h. The reaction mixture was dispersed in distilled water (50 ml) and washed with CH 2 Cl 2 (3×50 ml) extraction. The combined organic layers were purified by Na 2 SO 4 The residue was dissolved in MeOH (5 ml) and stirred at room temperature with NH 4 F (300 mg; 8.1 mmol) was stirred for 24 h. The solvent was removed under reduced pressure. The reaction mixture was concentrated under reduced pressure and 2 O and CH 2 Cl 2 The organic layer was separated and purified by Na 2 SO 4 After concentration, the crude product was purified by flash column chromatography (ethyl acetate / methanol, 100:0 to 98:2) to obtain compound dG-5 in the form of a white powder. Compound dG-5 (123 mg; 0.28 mmol) and proton sponge (75.8 mg; 0.35 mmol) were mixed in a vacuum desiccator over P 2 O 5 Dry overnight and then dissolve in trimethyl phosphate (600 ml). Freshly distilled POCl was then added dropwise at 0 °C. 3(40 μl; 0.35 mmol) and the mixture was stirred at 0°C for 2 h. Subsequently, a well-vortexed mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 ml; 2.31 mmol) in anhydrous DMF (2.33 ml) was added in one portion at room temperature and stirred for 30 min. Triethylammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 ml) was then added and the mixture was stirred at room temperature for 1 h. Conc. NH 4 OH (15 ml) and stirred at room temperature overnight. The resulting mixture was concentrated under vacuum and the residue was diluted with 5 ml of water. The crude mixture was 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 was further purified by reverse phase HPLC to give (L) 3'-O-azidomethyl-dGTP (compound dG-6).

[0320] 6.1.3. Synthesis of (L) 3'-O-azidomethyl-dCTP

[0321] 6.1.3.1 Scheme 3. (L) 3'-ON 3 Synthesis of -dCTP

[0322]

[0323] The preparation procedure of (L)3'-O-azidomethyl-dCTP (Compound dC-5) is similar to the synthetic scheme outlined above for (L)3'-O-azidomethyl-dATP (5 steps). The starting material used for the synthesis of (L)3'-O-azidomethyl-dCTP is β-L-deoxycytidine (Chemgenes).

[0324] 6.1.4. Predictive Synthesis of (L) 3'-O-Azidomethyl-dTTP

[0325] 6.1.4.1 Scheme 4. (L) 3'-ON 3 Synthesis of -dTTP

[0326]

[0327] To a stirred mixture of 'starting material' (β-L-deoxythymidine) (1.3 g; 3.87 mmol) and imidazole (693 mg; 9.51 mmol) in anhydrous DMF (21.0 mL) was added tert-butyldimethylsilyl chloride (TBDMSCl) (765 mg; 4.92 mmol). The reaction mixture was stirred at room temperature for 20 h. After evaporation, the residue was purified by flash column chromatography using CH3 OH-CH 2 Cl 2 Purification was performed with 4% paraformaldehyde (1:20) as the eluent to obtain 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) were added acetic acid (5.5 ml) and acetic anhydride (17.6 ml). The reaction mixture was stirred at room temperature for 48 h. Saturated NaHCO 3 solution (100 ml) and the aqueous layer was washed with CH 2 Cl 2 The combined organic extracts were washed with saturated NaHCO 3 solution (100 ml) and washed with Na 2 SO 4 After concentration, the residue was purified by flash column chromatography (hexane / ethyl acetate, 1:1 to 1:4) to give compound dT-2 in the form of a white powder. Under nitrogen, compound dT-2 (400 mg; 0.76 mmol) was added to dry CH 2 Cl 2 To a stirred solution of 1% cyclohexene (400 μl) and SO 2 Cl 2 (155 μl; 1.91 mmol, redistilled). The reaction mixture was stirred at 0°C for 2 h. The solvent was removed first under reduced pressure and then under a high vacuum pump for 10 min. The residue was dissolved in dry DMF (5 ml) and stirred at room temperature with NaN 3 (400 mg; 6.6 mmol) was reacted for 3 h. The reaction mixture was dispersed in distilled water (50 ml) and washed with CH 2 Cl 2 (3×50 ml) extraction. The combined organic layers were purified by Na 2 SO 4 The residue was dissolved in MeOH (5 ml) and stirred at room temperature with NH 4 F (300 mg; 8.1 mmol) was stirred for 24 h. The solvent was removed under reduced pressure. The reaction mixture was concentrated under reduced pressure and 2 O and CH 2 Cl 2 The organic layer was separated and purified by Na 2 SO 4After concentration, the crude product was purified by flash column chromatography (ethyl acetate / methanol, 100:0 to 98:2) to obtain compound dT-3 in the form of a white powder. Compound dT-3 (123 mg; 0.3 mmol) and proton sponge (75.8 mg; 0.35 mmol) were mixed in a vacuum desiccator over P 2 O 5 Dry overnight and then dissolve in trimethyl phosphate (600 μl). Freshly distilled POCl was then added dropwise at 0 °C. 3 (40 μl; 0.35 mmol) and the mixture was stirred at 0°C for 2 h. Subsequently, a well-vortexed mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 ml; 2.31 mmol) in anhydrous DMF (2.33 ml) was added all at once and stirred for 30 min at room temperature. Triethylammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 ml) was then added and the mixture was stirred at room temperature for 1 h. Conc. NH 4 OH (15 ml) and stirred at room temperature overnight. The resulting mixture was concentrated under vacuum and the residue was diluted with 5 ml of water. The crude mixture was 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 was further purified by reverse phase HPLC to obtain (L) 3'-O-azidomethyl-dTTP (compound dT-4).

[0328] 6.2. Example 2. (L) Synthesis of 3'-O-R'-dNTP

[0329] Having reviewed the exemplary methods provided in this application, one skilled in the art will be able to readily adapt these methods to produce compounds with various R' groups protecting 3'O. Specifically, allyl and nitrobenzyl analogs of the described (L)-3'-O-azidomethyl-dNTPs are contemplated, utilizing allyl bromide and 2-nitrobenzyl bromide, respectively, instead of the acetic acid / acetic anhydride steps in the above reaction schemes 1-4. Specific 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 these documents (including supporting information) are incorporated into this application by reference.

[0330] 6.3. Example 3. (L) Synthesis of 3'-O-azidomethyl-dNTP-label

[0331] 6.3.1. Synthesis of (L) 3'-O-azidomethyl-dATP-ROX

[0332] 6.3.1.1 Scheme 5. NH 2 -(L)3'-ON 3 -dATP synthesis

[0333]

[0334] 6.3.1.2 Scheme 6. (L) 3'-ON 3 Synthesis of -dATP-ROX

[0335]

[0336] Azide-ROX compounds (ROX- 3 -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 Application US20060160081A1) was dissolved in DMF (300 μl) and 1 M NaHCO 3 A solution of ROX NHS (N-hydroxysuccinimide) ester (Invitrogen) (0.013 mmol) in DMF (400 μl) was slowly added to the above reaction mixture, and then stirred for 5 h at room temperature in the absence of light. The crude product was analyzed on a preparative silica gel TLC plate (CHCl 3 / CH 3 OH, 1:4).

[0337] L-3'-O-azidomethyl-dATP-ROX compound (L-3'-O'-N 3 -dATP-ROX). 3 -Linker was added to a stirred solution of DSC (N,N'-disuccinimidyl carbonate) (3.4 mg, 13.2 μmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 μmol) in dry DMF (2 ml). The reaction mixture was stirred at room temperature for 2 h. TLC showed that ROX-N 3 -The linker is completely converted to the compound ROX-N 3 - linker NHS ester, which is used directly in the reaction with NaHCO 3 / Na 2 CO 3The reaction mixture was stirred at room temperature for 3 h in the absence of light. The reaction mixture was then separated by preparative silica gel TLC plates (CH 3 OH / CH 2 Cl 2 The crude product was further purified on reverse phase HPLC to obtain L-3'-O-azidomethyl-dATP-ROX (L-3'-ON 3 -dATP-ROX).

[0338] 6.3.2. (L) Synthesis of 3'-O-azidomethyl-dGTP-Cy5

[0339] 6.3.2.1 Scheme 7. NH 2 -(L)3'-ON 3 Synthesis of -dGTP

[0340]

[0341]

[0342] 6.3.2.2 Scheme 8. (L) 3'-ON 3 -dGTP-Cy5

[0343]

[0344] Azide-Cy5 compounds (Cy5-N 3 -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 Application US20060160081A1) was dissolved in DMF (300 μl) and 1 M NaHCO 3 A solution of Cy5 NHS (N-hydroxysuccinimide) ester (Invitrogen) (0.013 mmol) in DMF (400 μl) was slowly added to the above reaction mixture, and then stirred for 5 h at room temperature in the absence of light. The crude product was purified by preparative silica gel TLC plate (CHCl 3 / CH 3 OH, 1:4).

[0345] L-3'-O-azidomethyl-dGTP-Cy5 compound (L-3'-ON 3-dGTP-Cy5). 3 -Linker was added to a stirred solution of DSC (N,N'-disuccinimidyl carbonate) (3.4 mg, 13.2 μmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 μmol) in dry DMF (2 ml). The reaction mixture was stirred at room temperature for 2 h. TLC showed that Cy5-N 3 -The linker is completely converted into the compound Cy5-N 3 - linker NHS ester, which is used directly in the reaction with NaHCO 3 / Na 2 CO 3 The reaction mixture was stirred at room temperature for 3 h in the absence of light. The reaction mixture was separated by preparative silica gel TLC plate (CH 3 OH / CH 2 Cl 2 The crude product was further purified on reverse phase HPLC to obtain L-3'-O-azidomethyl-dGTP-Cy5 (L-3'-ON 3 -dGTP-Cy5).

[0346] 6.3.3. Synthesis of (L) 3'-O-azidomethyl-dCTP-Bodipy-FL-510

[0347] 6.3.3.1 Scheme 9. NH 2 -(L)3'-ON 3 Synthesis of -dCTP

[0348]

[0349] 6.3.3.2 Scheme 10. (L) 3'-ON 3 Synthesis of -dCTP-Bodipy-FL-510

[0350]

[0351] Azido-Bodipy-FL-510 (compound BODIPY-FL-510-N 3-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 Application US20060160081A1) was dissolved in DMF (300 μl) and 1 M NaHCO 3 Aqueous solution (100 μl) was added. A solution of Bodipy-FL-510 NHS (N-hydroxysuccinimide) ester (Invitrogen) (5.0 mg, 0.013 mmol) in DMF (400 μl) was slowly added to the above reaction mixture, and then stirred for 5 h at room temperature in the absence of light. The crude product was analyzed on a preparative silica gel TLC plate (CHCl 3 / CH 3 OH, 1:4) to obtain BODIPY-FL-510-N 3 -Connector.

[0352] L-3'-O-azidomethyl-dCTP-Bodipy-FL-510 (compound L-3'-ON 3 -dCTP-Bodipy-FL-510). To BODIPY-FL-510-N 3 -Linker DSC (N,N'-disuccinimidyl carbonate) (3.4 mg, 13.2 μmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 μmol) were added to a stirred solution in dry DMF (2 ml). The reaction mixture was stirred at room temperature for 2 h. TLC showed that BODIPY-FL-510-N 3 -The linker is completely converted to compound BODIPY-FL-510-N 3 - linker NHS ester, which is used directly in the reaction with NaHCO 3 / Na 2 CO 3 The reaction mixture was stirred at room temperature for 3 h in the absence of light. The reaction mixture was separated by preparative silica gel TLC plate (CH 3 OH / CH 2 Cl 2 The crude product was further purified on reverse phase HPLC to give L-3'-O-azidomethyl-dCTP-Bodipy-FL-510.

[0353] 6.3.4. (L) Synthesis of 3'-O-azidomethyl-dUTP-R6G

[0354] 6.3.4.1 Scheme 11. NH 2 -(L)3'-ON 3 Synthesis of -dUTP

[0355]

[0356] 6.3.4.2 Scheme 12. (L) 3'-ON 3 Synthesis of -dUTP-R6G

[0357]

[0358] Azide-R6G (compound R6G-N 3 -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 Application US20060160081A1) was dissolved in DMF (300 μl) and 1 M NaHCO 3 A solution of R6G NHS (N-hydroxysuccinimide) ester (Invitrogen) (0.013 mmol) in DMF (400 μl) was slowly added to the above reaction mixture, and then stirred for 5 h at room temperature in the absence of light. The crude product was purified by preparative silica gel TLC plate (CHCl 3 / CH 3 OH, 1:4).

[0359] L-3'-O-azidomethyl-dUTP-R6G. 3 -linker was added to a stirred solution of dry DMF (2 ml) with DSC (N,N'-disuccinimidyl carbonate) (3.4 mg, 13.2 μmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 μmol). The reaction mixture was stirred at room temperature for 2 h. TLC showed that R6G-N 3 -The linker is completely converted to compound R6G-N 3 - linker NHS ester, which is used directly in the reaction with NaHCO 3 / Na 2 CO 3The reaction mixture was stirred at room temperature for 3 h in the absence of light. The reaction mixture was then separated by preparative silica gel TLC plates (CH 3 OH / CH 2 Cl 2 The crude product was further purified on reverse phase HPLC to obtain L-3'-O-azidomethyl-dUTP-R6G (L-3'-ON 3 -dUTP-R6G).

[0360] 6.4. Example 4. (L) Synthesis of 3'-O-allyl-dNTP-allyl-label

[0361] Reviewing the exemplary methods provided in this application, one skilled in the art will be able to readily adapt the provided methods to produce compounds with various R' groups protecting 3'O and alternative cleavable linkers (e.g., incorporating the allyl-fluorophore linker reported in Ju J. et al. 2006, PNAS, Vol. 103, No. 52) according to the following scheme. Specifically, it is contemplated that the described (L)-3'-O-azidomethyl-dNTP-NH 2 Allyl analogs of the present invention are prepared by using allyl bromide instead of the acetic acid / acetic anhydride steps in the above reaction schemes 5, 7, 9 and 11. Specific 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 into this application by reference.

[0362]

[0363] 1. Synthesis of Allyl-Linkers

[0364]

[0365] 6.5. Example 5. Synthesis of Mirror Image Polymerase

[0366] The amino acid sequence of 9°N polymerase was divided into multiple fragments. Each polypeptide fragment was synthesized using a solid phase peptide synthesis method (Fmoc-SPPS) based on a strategy using 9-fluorenylmethoxycarbonyl (Fmoc) as a protecting group. Once the polypeptide fragments were synthesized, they were separated and purified using semi-preparative grade reversed phase high performance liquid chromatography (RP-HPLC). The separated polypeptide fragments were connected using a native chemical ligation method from the C-terminus to the N-terminus. After the chemical ligation reaction was completed, the target product was isolated using semi-preparative grade RP-HPLC.

[0367] The synthesized SPI polymerase was folded and renatured. Circular dichroism and mass spectrometry were used to confirm that the SPI polymerase was correctly folded.

[0368] 6.6. Example 6. Activity of Mirror Image Polymerase

[0369] The mirror image 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 image polymerase is measured based on the yield of polynucleotide fragments complementary to the template or using a double-stranded DNA intercalating dye (e.g., EvaGreen dye) and measuring the increase in fluorescence.

[0370] 6.7. Example 7. Mirror-image polymerase chain reaction

[0371] Mirror image polymerase was 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 initial cycle consisted of 5 min at 95°C, 5 min at 50°C (during which polymerase and BSA additions were performed) and 5 min at 70°C. The segments of each subsequent PCR cycle were 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 of the 100 μl volume was removed and subjected to agarose gel electrophoresis in the presence of ethidium bromide to quantify the amplification of the template sequence. The reaction produced polynucleotide fragments complementary to the template.

[0372] 6.8. Example 8. Use of (L)-3'-OR-dNTP, (L)-3'-OR-dNTPs-R-label, (L)-ddNTPs-R 2 -labeling and -specific single-base extension of L-primers by D-polymerase, deprotection, and reinitiation of extension to demonstrate the general procedure of sequencing by synthesis (SBS)

[0373] To obtain de novo DNA sequencing data for L-primer / L-DNA template immobilized on a solid surface, first, a 3'-ON 3 -dCTP (3 μM), 3'-ON 3 -dTTP (3 μM), 3'-ON 3 -dATP (3 μM) and 3'-ON 3 Solution A consisted of -dGTP (0.5 μM) and ddCTP- 3-Bodipy-FL-510(50nM), ddUTP-N 3 -R6G (100nM), ddATP-N 3 -ROX (200 nM) and ddGTP-N 3 -Cy5 (100 nM) was used to verify the accurate and specific single base extension. For example, in addition to the modified L-nucleotide reversible terminator, 60 pmol of self-priming DNA template, 1X Thermopol II reaction buffer, 40 nmol MnCl 2 The reaction was performed by incubating at 94°C for 5 min, at 4°C for 5 min, and at 65°C for 20 min. The extension products were then analyzed by fluorescence-based gel electrophoresis (and additionally, MALDI-TOF MS can be used as an alternative) to confirm the specific incorporation of the correct nucleotides. 3 -dNTP and ddNTP-N 3 -cleavage of the DNA extension product of the fluorophore, the DNA product is resuspended in 50 μl of 100 mM TCEP solution (pH 9.0) at 65°C for 15 min and then analyzed by gel electrophoresis or MALDI-TOF MS to confirm the cleavage of the R-label / protecting group, thereby allowing the reinitiation of extension of the next base. A complete single cycle of SBS (extension, label detection and cleavage) is described above.

[0374] Typically, a 'solution A' consisting of four L-3'-O-R'-dNTPs (each with dATP, dTTP, dGTP or dCTP) and a 'solution A' consisting of four L-ddNTP-R 2 -labeled (each with ddATP, ddTTP, ddGTP or ddCTP). 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 sequencing by synthesis (SBS). During the SBS cycle, the mirror polymerase uses a 3′-capped nucleotide reversible terminator (L-3'-O-R'-dNTP) and a cleavable fluorescent dideoxynucleotide (L-ddNTPs-R 2 The complementary sequence to the L-polynucleotide is synthesized by combining the L-polynucleotide with a marker. The signal from the marker specific for ddATP, ddTTP, ddGTP or ddCTP is used to detect the signal from the marker specific for ddNTP (L-ddNTPs-R 2After the signal is detected, the 3′-OH capping group (R′ group) from the incorporated nucleotide reversible terminator (L-3′-O-R′-dNTP) and the 3′-OH capping group (R′ group) from the ddNTP (L-ddNTPs-R 2 The marker cleavage of the DNA product terminated by the L-label (-label) is used to restart the polymerase reaction. Since each marker conjugated with dATP, dTTP, dGTP or dCTP is unique, the fluorescent signal from the marker indicates the nucleotide corresponding to the termination site. Based on the signal, the sequence of the L-polynucleotide template is determined.

[0375] The L-polynucleotide template also uses four L-3'-O-R'-dNTP-R 2 -labeled (each with dATP, dTTP, dGTP or dCTP) sequencing. 2 -label (each with dATP, dTTP, dGTP or dCTP), L-primer, D-polymerase and buffer are mixed under similar reaction conditions as described above. The mixture is incubated through 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 includes a chemical portion (R') that caps the 3'-OH and a chemically cleavable linker (R 2 ) is tethered to the base. After incorporation, the fluorescent signal from the label is detected, and then the 3'-capping part (R') and the label on the reaction product are cleaved to restart the polymerase reaction. Since each label conjugated with 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.

[0376] 6.9. Example 9. Sequencing of L-polynucleotides by a method similar to Sanger sequencing

[0377] L-polynucleotide template uses four L-ddNTP-R 2 -labeled (each with ddATP, ddTTP, ddGTP or ddCTP) for sequencing (similar to Sanger sequencing). 2 -label was mixed with L-polynucleotide template, L-primer, four L-dNTPs (L-dATP, L-dGTP, L-dCTP and L-dTTP), D-polymerase and buffer under similar reaction conditions as described above. In the mixture, L-ddNTP-R 2- The marker is added to the mixture in much smaller amounts. PCR (cycle sequencing - generation of a ladder of DNA fragments) is performed with the mixture. The replication products are separated by size using gel electrophoresis and the L-ddNTPs-R from the incorporated replication products are detected. 2 -The signal of the label. Since each label conjugated with 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 the fragment mobility (based on size), the sequence of the L-polynucleotide template is determined.

[0378] 6.10. Example 10. Complete Synthesis of (L)-3'-O-Azidomethyl-dNTP

[0379] As shown in Schemes 13–16, four target molecules and their synthetic pathways were designed. L-3'-O-azidomethyl-dTTP (dT-4) and L-3'-O-azidomethyl-dCTP (dC-5) were prepared and 1 H. 31 P NMR and HRMS were used for characterization. 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.

[0380] Materials and methods

[0381] Unless otherwise stated, all solvents and reagents were of reagent grade, purchased commercially and used without further purification. All chemicals were purchased from Sigma-Aldrich, Fisher Scientific, TCI, etc. 1 H NMR was performed on a Bruker Ascend from Chapman University TM (400 MHz) spectrometer and recorded according to CDCl 3 (7.26ppm) or D 2 O is reported as parts per million (ppm). Data are reported as follows: (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets, J = coupling constant in Hz, integrated). 31 P NMR spectra were obtained from a Bruker Ascend TM(121.4MHz) spectrometer. High-resolution mass spectra (HRMS) were obtained from Chapman University School of Pharmacy and the Analytical Chemistry Instrumentation Facility at the University of California, Riverside. Starting materials β-L-deoxythymidine, β-L-deoxycytidine, β-L-deoxyadenosine, and β-L-deoxyguanosine were purchased from Chemgenes. Analytical (Polaris180A C18-A, 4.6x250mm, 5um) and semi-preparative (Polaris 180A C18-A, 4.6x 250mm, 5um) HPLC columns were purchased from Agilent. The 3'-O-modified nucleotides were purified by reverse phase HPLC on a 4.6x 250mm C18 column (Polaris), mobile phase: A, 25mM TEAB buffer in water; B, 25mM TEAB buffer in acetonitrile. Elution was performed under isocratic conditions as described in each procedure.

[0382] 6.10.1. (L) Synthesis of 3'-O-azidomethyl-dTTP

[0383] 6.10.1.1 Scheme 13. (L) 3'-ON 3 - Synthesis of dTTP (dT-4)

[0384]

[0385] Experimental Procedure:

[0386] 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) at 0°C under nitrogen. After stirring at room temperature for 3 h, ice-cold water was added to the solution and extracted with EtOAc (2×50 mL). The combined organic layers were purified by Na 2 SO 4 The residue was dissolved in CH3OH and silica gel was added. The solution was dried under reduced pressure until dry. The residue was purified by column chromatography (2:98 to 5:95, CH 3 OH–CH 2 Cl 2 ) to obtain dT-1 (1.8 g, 82%) as a white solid. f 0.35(1:19CH 3 OH–CH 2 Cl 2 ). The product was confirmed by TLC.

[0387] Synthesis of dT-2: 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. At 0 °C, saturated NaHCO 3 The solution was stirred for 30 min, and the aqueous layer was washed with CH 2 Cl 2 (2×100 ml) extraction. The combined organic layers were purified by Na 2 SO 4 Dry, filter and concentrate. The resulting residue was purified by flash column chromatography (1:1, hexanes-EtOAc) to give dT-2 (2.0 g, 90%) as a light yellow syrup. f 0.55 (1:1 hexane-EtOAc); 1 HNMR (400MHz, CDCL 3 ):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,CH 2 S),4.61(d,1H,J gem =11.8Hz,CH 2 S),4.47(app dt,1H,J 3’,2’b =5.9Hz,J 3’,2’a =1.9Hz,H-3'),4.12–4.08(m,1H,H-4'),3.89(dd,1H,J 5’a,4’ =2.6Hz,J gem =11.3Hz,H-5'a),3.80(dd,1H,J 5’a,4’ =2.9Hz,J gem =11.3Hz,H-5'b),2.41(ddd,1H,J 2’a,3’ =1.9Hz,J 2’a,1’ =5.6Hz,J gem =13.6Hz,H-2'a),2.16(s,3H,SCH 3 ),1.99(ddd,1H,J 2’b,3’ =5.9Hz,J 2’b,1’ =8.6Hz,J gem =13.6Hz,H-2'b),1.92(d,3H, 4 J=1.1Hz,CH3 ),0.93(s,9H,(CH 3 ) 3 CSi),0.12(s,6H,(CH 3 ) 2 Si)); HRMS (ESI) m / z [M+H] + Calculated as C 18 H 33 N 2 O 5 SSi 417.1879; measured value 417.1881.

[0388]

[0389] Synthesis of dT-3: dT-2 (2.0 g, 4.80 mmol) was added to dry CH 2 Cl 2 To a stirred solution of 1% cyclohexene (2.1 mL) and SO 2 Cl 2 (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 stirred with NaN at room temperature. 3 (1.88 g, 28.8 mmol) was reacted 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 purified by Na 2 SO 4 The residue was dissolved in CH 3 CN (10 mL) and reacted with 2M HCl (2-3 mL) at 0 °C for 5 h. Saturated Na 2 CO 3 solution and with CH 2 Cl 2 (2 × 50 mL) extraction, in Na 2 SO 4 The organic layer was washed with water and the organic layer was concentrated over Na 2 SO 4 The residue was purified by flash column chromatography (hexane / ethyl acetate, 3:7 to 1:4) to afford 3 as a white powder. f 0.20 (1:4 hexane-EtOAc); 1 HNMR (400MHz, CDCL 3 ):δ8.63(s,1H,NH),7.38(d,1H, 4J=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,CH 2 N 3 ),4.70(d,1H,J gem =9.0Hz,CH 2 N 3 ),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, 4 J=1.1Hz,CH 3 ); HRMS (ESI) m / z [M+H] + Calculated as C 11 H 16 N 5 O 5 S298.1151; measured value 298.1144

[0390]

[0391] Synthesis of dT-4: dT-3 (120 mg, 0.403 mmol) was dried in a vacuum dryer over P 2 O 5 Dry overnight. To a solution of dT-3 in trimethyl phosphate (5 mL) was added POCl dropwise at 0 °C. 3 (94.3uL, 1.00mmol). The mixture was stirred at 0°C for 2 hours, then a mixture of tributylammonium pyrophosphate (552mg) and tributylamine (738uL, 3.10mmol) in anhydrous DMF (2mL) was added and vortexed thoroughly. The mixture was stirred at room temperature for 1 hour, then 0.1M triethylammonium bicarbonate buffer (TEAB buffer, pH 8.5, 20ml) 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 10ml of water. The crude mixture was washed with CH 2 Cl 2(2×10 mL) was extracted 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; 0.1–0.8 M). Fractions with the product (0.3 M–0.4 M) were collected and concentrated under reduced pressure. The residue was washed with ddH 2 The mixture was diluted with 1% TEAB 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. f 0.28 (1:2TEAB–ACN); 1 H NMR (400 MHz, D 2 O):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,CH 2 N 3 ),4.69(d,1H,J gem =8.8Hz,CH 2 N 3 ),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,CH 3 ); 31 PNMR (121.4MHz, D 2 O): δ–10.95(bs,1P),–11.75(d,J=20.0Hz,1P),–23.36(bs,1P); HRMS(ESI)m / z[M–H] – Calculated as C 11 H 17 N 5 O 14 P3 – 535.9990; measured value 535.9993.

[0392] dT-4:

[0393] 6.10.2. Synthesis of L-3'-O-azidomethyl-dCTP:

[0394] 6.10.2.1 Scheme 14. L-3'-ON 3 Synthesis of -dCTP(dC-5)

[0395]

[0396] Synthesis of dC-1: Under nitrogen, β-L-deoxycytidine (1 g, 4.40 mmol) was co-evaporated with anhydrous pyridine (2×10 mL) and then dissolved in anhydrous pyridine (15 mL). Trimethylsilyl chloride (TMSCl, 2.79 mL, 22.0 mmol) was slowly added and the mixture was stirred at room temperature for 1 hour, followed by addition of benzoyl chloride (2.56 mL, 22.0 mmol) 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. Then, concentrated ammonia solution (15 mL) was added and the solution was stirred for another 1 hour while warming to room temperature. The solvent was evaporated under reduced pressure and the resulting crude product was purified by column chromatography (19:1, CH 2 Cl 2 –CH 3 OH) to obtain dC-1 (520 mg, 36%) as a white solid. f 0.35(1:19CH 3 OH–CH 2 Cl 2 ).

[0397] Synthesis of dC-2: At 0°C, imidazole (172 mg, 2.53 mmol) and tert-butyldimethylsilyl chloride (204 mg, 1.35 mmol) were added to a solution of dC-1 (280 mg, 0.84 mmol) in anhydrous DMF (5.0 mL). The solution was stirred at 0°C for 3 hours under nitrogen. After the reaction was completed (TLC monitoring), cold water (10 mL) was added and extracted with EtOAc (3×20 mL). The combined organic layers were purified by Na 2 SO 4 Dry, filter and concentrate. The residue was purified by column chromatography (19:1 to 9:1, CH 2 Cl 2 –CH 3OH) to give dC-2 (280 mg, 74%) as a colorless oil. f 0.32(1:19CH 3 OH–CH 2 Cl 2 ).

[0398] 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. At 0 °C, saturated NaHCO 3 The solution was stirred for 30 min, and the aqueous layer was washed with CH 2 Cl 2 (2 × 30 ml) extraction. The combined organic extracts were purified by Na 2 SO 4 Dry, filter and concentrate. The crude product was purified by flash column chromatography (1:1, hexane-EtOAc) to give dC-3 (163 mg, 60%) as a white powder. f 0.22 (1:1 hexane-EtOAc); 1 HNMR (400MHz, CDCL 3 ): 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,CH 2 S),4.61(d,1H,J gem =11.7Hz,CH 2 S),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,Jgem =13.7Hz,H-2'a),2.21–2.13(m,4H,H-2'b,SCH 3 ),0.95(s,9H,(CH 3 ) 3 CSi); 0.15(s,3H,CH 3 Si), 0.14(s, 3H, CH 3 Si); HRMS (ESI) m / z [M+H] + Calculated as C 24 H 36 N 3 O 5 SSi 506.2145; measured value 506.2150.

[0399] Synthesis of dC-4. dC-3 (220 mg, 0.67 mmol) was added to dry CH 2 Cl 2 To a stirred solution of 1% cyclohexene (6 mL) was added cyclohexene (200 uL) and 2 Cl 2 SO 2 Cl 2 (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 3 (169 mg, 2.61 mmol) for 3 hours. The reaction mixture was dispersed in cold distilled water (30 mL) and extracted with EtOAc (2×30 mL). The combined organic extracts were purified by Na 2 SO 4 The residue was removed by TBDMS reaction by 2M HCl at room temperature for 1-2 h. The mixture was washed with saturated NaHCO 3 The solution was neutralized and diluted with EtOAc. The organic layer was washed with Na 2 SO 4 Dry and concentrate under reduced pressure. The residue was purified by flash column chromatography (1:2, hexanes-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 (400 MHz, CDCL 3): 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,CH 2 N 3 ),4.68(d,1H,J gem =9.2Hz,CH 2 N 3 ),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] + Calculated as C 17 H 19 N 6 O 5 387.1417; measured value 387.1426.

[0400] Synthesis of dC-5: dC-4 (100 mg, 0.259 mmol) was dried in a vacuum dryer over P 2 O 5 Dry overnight. To a solution of dC-4 in trimethyl phosphate (5 mL) was added POCl dropwise at 0 °C. 3The mixture was stirred at 0 ° C for 2 hours, and then a mixture of tributylammonium pyrophosphate (355mg, 0.647mmol) and tributylamine (473.8uL, 1.99mmol) in anhydrous DMF (2mL) was added to fully vortex. The mixture was stirred at room temperature for 1.5 hours, and then 0.1M triethylammonium bicarbonate buffer (TEAB buffer, pH 8.5, 0.1M, 15ml) was added, and the mixture was stirred at room temperature for 1 hour. Then concentrated ammonium hydroxide (10mL) was added to the mixture and stirred at room temperature overnight. The resulting mixture was concentrated under reduced pressure and the residue was diluted with 30ml of water. The crude mixture was washed with CH 2 Cl 2 (2×20 mL) was extracted 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 with the product (0.3 M–0.4 M) were collected and concentrated under reduced pressure. The residue was washed with ddH 2 The mixture was diluted with 1% TEAB 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. f 0.43 (1:2TEAB–ACN); 1 H NMR (400 MHz, D 2 O): 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,CH 2 N 3 ),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[M–H] – Calculated as C 10 H 16 N 5 O 13 P 3 – 520.9994; measured value 520.9990.

[0401] 6.10.3.L-3'-O-azidomethyl-dATP synthesis:

[0402] 6.10.3.1 Scheme 15. L-3'-ON 3 - Synthesis of dATP (dA-5)

[0403]

[0404] Experimental Procedure:

[0405] Synthesis of dA-1: β-L-deoxyadenosine (2.0 g, 7.96 mmol) was co-evaporated 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 trimethylsilyl chloride (TMSCl, 5.06 mL, 39.8 mmol) was added dropwise via a syringe. The mixture was stirred at 0 ° C for 1 hour. Benzoyl chloride (4.62 mL, 39.8 mmol) was added dropwise to the solution via a syringe and the ice bath was removed. After stirring at room temperature for 3 hours, 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 CH 3 OH and silica gel was added. The mixture was dried under reduced pressure. The residue was purified by column chromatography (49:1 to 19:1, CH 2 Cl 2 –CH 3 OH) to afford dA-1 (2.12 g, 75%) as a white solid.

[0406] 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 hours. After the reaction was complete, cold water (50 mL) was added and the mixture was extracted with EtOAc (3×50 mL). The combined organic layers were purified by anhydrous Na 2 SO 4 Dried, filtered and concentrated. The resulting residue was purified by column chromatography (1:2, hexanes-EtOAc to 100% EtOAc) to give compound dA-2 (1.92 g, 69%) as a colorless oil.

[0407] 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 hours, saturated NaHCO 3 The solution was stirred for 30 min. The mixture was washed with CH 2 Cl 2 (2×100 ml) extraction. The combined organic layers were purified by Na 2 SO 4 Dry, filter and concentrate. The residue was purified by flash column chromatography (1:1, hexanes-EtOAc) to afford dA-3 (1.27 g, 63%) as a white foam. f 0.28 (1:7 hexane-EtOAc); 1 H NMR (400 MHz, CDCL 3 ): 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,CH 2 S,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,SCH 3 ),0.91(s,9H,(CH 3 ) 3 CSi); 0.10(s,6H,(CH 3 ) 2 Si); HRMS (ESI) m / z [M+H]+ Calculated as C 25 H 36 N 5 O 4 SSi 530.2157; measured value 530.2132.

[0408] 6.10.4.L-3'-O-azidomethyl-dGTP Synthesis:

[0409] 6.10.4.1 Scheme 16. L-3'-ON 3 Synthesis of -dGTP(dG-6)

[0410]

[0411] Experimental Procedure:

[0412] Synthesis of dG-1: β-L-deoxyguanosine (2 g, 7.48 mmol) was co-evaporated (2×12 mL), then 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 a syringe. The ice bath was then removed, and the mixture was stirred for 1 hour. The solution was cooled to 0° C., and isobutyric anhydride (6.2 mL, 37.4 mmol) was added dropwise via a syringe. The ice bath was removed, and the resulting solution was stirred at room temperature for 3 hours. After stirring for 3 hours, the mixture was cooled to 0° C. and ice-cold water (5 mL) was slowly added and stirred for 15 min, followed by the addition of concentrated ammonia solution (10 mL) to a final concentration of 2.5 M ammonia. The mixture was stirred on an ice bath for 1 hour, then evaporated to dryness. The residue was redissolved in CH 3 OH and add silica gel and concentrate until dryness. The mixture is concentrated until dryness. The resulting residue is purified by column chromatography (19:1 to 9:1, CH 2 Cl 2 –CH 3 OH) to afford dG-1 (1.85 g, 73%) as a white solid.

[0413] Synthesis of dG-2: At 0 ° C, under nitrogen, 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), 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 washed with anhydrous Na 2 SO 4 Dry, filter and concentrate. The residue was purified by column chromatography (19:1 to 9:1, CH2 Cl 2 –CH 3 OH) to afford dG-2 (1.9 g, 79%) as a white foam.

[0414] Synthesis of dG-3: To a stirred solution of dG-2 (1.7 g; 3.76 mmol) in DMSO (12 ml) were added acetic acid (6 ml) and acetic anhydride (18 ml). After stirring at room temperature for 48 h, saturated NaHCO 3 The solution was stirred for 30 min, and the aqueous layer was extracted with EtOAc (2×100 ml). The combined organic layers were purified by Na 2 SO 4 Dry, filter and concentrate. The crude product was purified by flash column chromatography (1:1 to 1:3, hexanes-EtOAc) to afford dG-3 (1.2 g; 65%) as a light yellow foam. f 0.26 (1:4 hexane-EtOAc); 1 H NMR (400 MHz, CDCL 3 ): 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,CH 2 S,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(CH 3 ) 2 ),2.57–2.46(m,2H,H-2'a,H-2'b),2.15(s,3H,SCH 3 ),1.25(app t,6H,J=7.0Hz,COCH(CH 3 ) 2 ),0.89(s,9H,(CH 3 ) 3 CSi); 0.08(s,3H,(CH 3 ) 2 Si), 0.07(s, 3H, (CH 3 ) 2 Si); HRMS (ESI) m / z [M+H] + Calculated as C 22 H 38 N 5 O 5SSi512.2363; measured value 512.2360.

[0415] 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 hours, the solvent was removed under high vacuum. The residue was diluted with EtOAc and washed with 2M HCl and saturated NaHCO 3 The organic layer was washed with Na 2 SO 4 Dry, filter and concentrate. 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 light yellow powder. f 0.5 (1:1 hexane-EtOAc); 1 H NMR (400 MHz, CDCL 3 ): 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,CH 2 S,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(CH 3 ) 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,SCH 3 ),1.28(d,6H,J=6.8Hz,COCH(CH 3 )2 ),0.92(s,9H,(CH 3 ) 3 CSi); 0.11(s,3H,(CH 3 ) 2 Si), 0.10(s, 3H, (CH 3 ) 2 Si); HRMS (ESI) m / z [M+H] + Calculated as C 35 H 47 N 6 O 6 SSi 707.3047; measured value 707.3048.

[0416] Synthesis of dG-5: dG-4 (490 mg, 0.694 mmol) was added to dry CH at 0 °C. 2 Cl 2 Cyclohexene (2.1 mL) and distilled water in CH 2 Cl 2 1.0M SO 2 Cl 2 (1.39 mL, 1.39 mmol). After stirring at 0 °C for 1 hour, the volatiles were removed under reduced pressure. To a solution of the residue in dry DMF (14 mL) was added NaN 3 (271 mg, 4.61 mmol). After stirring at room temperature for 2 h, the reaction mixture was dispersed in distilled water (200 mL) and extracted with EtOAc (2×200 mL). The combined organic layers were purified by Na 2 SO 4 The mixture was dried, filtered and concentrated under reduced pressure. 2M HCl (7 mL) was added to a solution of the residue in acetonitrile (14 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 3 The organic layer was washed with water (2×50 mL) and the organic layer was washed with Na 2 SO 4 Dry, filter and concentrate under reduced pressure. The resulting residue is purified by flash column chromatography (1:2 to 0:1, hexanes-EtOAc) to afford dG-4 as a colorless oil. f 0.32 (1:4 hexane-EtOAc); 1 H NMR (400 MHz, CDCL 3): 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,CH 2 N 3 ),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(CH 3 ) 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] + Calculated as C 28 H 30 N 9 O 6 588.2219; measured value 588.2177.

[0417] 6.11. Example 11. (L) Complete Synthesis of 3'-O-Azidomethyl-dNTP-Labeled Intermediate

[0418] The synthesis of L-3'-O-azidomethyl-dNTP-FL was designed and divided into three parts: the synthesis of the linker (Scheme 17), the NH 2 -Synthesis of L-3'-O-azidomethyl-dNTP intermediates (Schemes 18-23), followed by coupling of the intermediates to fluorophores as described in Example 3 above.

[0419] The synthesis of the linker (Scheme 17) currently provides compound 5, referred to as intermediate linker-5, which is obtained by 1 The first NH 2-L-3'-O-azidomethyl-dNTP is NH 2 -Synthesis of L-3'-O-azidomethyl-dUTP (Scheme 18). 2 The synthesis of -L-3'-O-azidomethyl-dUTP was completed to step 3 and the 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).

[0420] Given the compatibility of fluorophore-attached dNTPs with 9°N DNA polymerase, we also designed NH 2 -(L)3'-ON 3 -7-deaza-dGTP (Scheme 22) and NH 2 -(L)3'-ON 3 -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 sugar (1-chloro-3,5-di-O-p-toluoyl-2-L-deoxyribofuranose) are commercially available.

[0421] 6.11.1. Scheme 17. Synthesis of linker

[0422]

[0423] Synthesis of Linker-1

[0424] 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 washed with CH 2 Cl 2 (250 mL) and washed with water. The aqueous layer was washed with CH 2 Cl 2 Wash twice (2×50 mL). The combined organic layers were washed with MgSO 4 Dried, filtered and concentrated. The residue was purified by column chromatography (hexane-EtOAc, 19:1 to 2:1) to obtain Linker-1 (4.6 g, 91%). f 0.54(100% CH 2 Cl 2 ); 1 H NMR (400 MHz, CDCL 3): 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,Ar H),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,OCH 2 CH 3 ),4.12–3.96(m,6H,OCH 2 CH 2 O,ArOCH 2 ),1.40(t,3H,J gem =7.2Hz,OCH 2 CH 3 ); HRMS

[0425]

[0426] Synthesis of Linker-2

[0427] 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 uL) at room temperature. The mixture was stirred at room temperature for 2 hours, and then 2% aqueous methanol (10 mL) was added. The mixture was stirred at room temperature for 30 min, and 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 obtain linker-2 (1.42 g, 45%). f 0.32 (2:1 hexane-EtOAc); 1 H NMR (400 MHz, CDCl 3 ): 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,OCH 2 CH 3 ),4.25(dd,1H,J=5.3,10.2Hz,ArOCH 2 ),4.17(dd,1H,J=5.3,10.2Hz,ArOCH 2),4.06–4.00(m,1H,OCH 2 ),3.91–3.73(m,3H,OCH 2 CH 2 OH),1.40(t,3H,J gem =7.2Hz,OCH 2 CH 3 ); HRMS (ESI) m / z [M–H] – Calculated as C 15 H 18 N 3 O 7 352.1236; measured value 352.1165

[0428]

[0429] Synthesis of Linker-3

[0430] 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 and acidified with 2N HCl (20 mL) and washed with CH 2 Cl 2 Extracted twice (2×50 mL). The combined organic layers were dried over MgSO 4 and filtered and concentrated to obtain Linker-3 (1.21 g, 94%). f 0.41(19:1EtOAc–CH 3 OH); 1 H NMR (400 MHz, CDCL 3 ): 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,1H,J=5.2Hz,CHN 3 ),4.26(dd,1H,J=5.2,10.1Hz,ArOCH 2 ),4.17(dd,1H,J=5.2,10.1Hz,ArOCH 2 ),4.08–4.01(m,1H,OCH 2 ),3.89–3.77(m,3H,OCH 2 CH 2 OH); HRMS (ESI) m / z [M–H] – Calculated as C11 H 12 N 3 O 5 266,0867; Measured value 266.0794

[0431]

[0432] Synthesis of Linker-4

[0433] 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 hours. After stirring for 4 hours, cold water (50 mL) was poured into the reaction mixture and the resulting mixture was washed with CH 2 Cl 2 The organic layer was discarded and the aqueous layer was acidified by adding 2N HCl (25 mL). The resulting mixture was washed with CH 2 Cl 2 (2×50 mL) and the collected organic layer was purified by Na 2 SO 4 Dry, filter and concentrate. The residue was purified by column chromatography (98:2, CH 2 Cl 2 –CH 3 OH) to obtain linker-4 (120 mg, 22%) as a light yellow oil. f 0.67 (1:1:0.5 EtOAc-hexane-AcOH); 1 H NMR (400 MHz, CDCl 3 ): 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,CHN 3 ),4.28–4.12(m,6H,ArOCH 2 ,OCH 2 CH 3 ,OCH 2 C=O),4.05(app dt,1H,J=4.1,11.4Hz,OCH 2 ),3.97–3.87(m,1H,OCH2 ),3.82(appt,2H,J=4.8Hz,OCH 2 ),1.29(t,3H,J=7.2Hz,OCH 2 CH 3 )HRMS(ESI)m / z[M–H] – Calculated as C 15 H 18 N 3 O 7 352.1236; measured value 352.1165.

[0434]

[0435] Synthesis of Linker-5

[0436] 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 washed with 1N Na 2 HPO 4 The solution (25 mL) was quenched. The mixture was washed with CH 2 Cl 2 Extraction was performed until no product was observed in the aqueous layer. The combined organic layers were purified by Na 2 SO 4 Dried, filtered and concentrated. The resulting residue was purified by column chromatography (1:1, hexane-EtOAc) to afford Linker-5 (42 mg, 25%) as a colorless oil. f 0.62 (1:1:0.5 EtOAc-hexane-AcOH); 1 H NMR (400 MHz, CDCl 3 ): 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,CHN 3 ),4.25–4.07(m,6H,ArOCH 2 ,OCH 2 CH 3 ,OCH 2C=O), 4.05–3.99 (m, 1H, OCH 2 ),3.89–3.82(m,1H,OCH 2 ),3.79(appt,2H,J=4.2Hz,OCH 2 ),3.70–3.62(m,2H,NCH 2 CH 2 N), 3.61–3.54 (m, 2H, NCH 2 CH 2 N), 1.28(t,3H,J=7.2Hz,OCH 2 CH 3 )

[0437]

[0438] 6.11.2. Intermediate NH 2 -Synthesis of (L) 3'-O-azidomethyl-dNTP:

[0439] 6.11.2.1 Scheme 18.NH 2 -(L)3'-ON 3 Synthesis of -dUTP

[0440]

[0441] Experimental Procedure:

[0442] Synthesis of dUTP-FL-1: β-L-deoxyuridine (2.4 g, 10.51 mmol, 1.0 eq) was suspended in methanol (30 mL) and stirred for 10 min. Iodine (8.0 g, 31.55 mmol, 3 eq) and silver nitrate (5.3 g, 31.55 mmol, 3 eq) were added later, and the reaction mixture was stirred at 40 °C for 3 hours. After the reaction was complete, the reaction mixture was filtered, and the filtrate was subjected to HPLC using MeOH / CH 2 Cl 2 1:19 to 1:9 column chromatography to obtain the compound dUTP-FL-1 (1.8 g, 48%) as a white solid. f 0.65(9:1DCM–MeOH), 1 HNMR (500 MHz, DMSO-d 6 )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).

[0443]

[0444] Synthesis of dUTP-FL-2: Compound 1 (400 g, 1.12 mmol, 1.0 eq) was dissolved in anhydrous DMF (20 mL). At 0 ° C, under nitrogen, imidazole (114.3 mg, 1.68 mmol, 1.5 eq) and tert-butyldimethylsilyl chloride (185.6 mg, 1.23 mmol, 1.1 eq) were added thereto. The reaction mixture was stirred at room temperature for 12 h. Cold water was then added and extracted with EtOAc (3×50 mL). The combined organic layers were washed with water and lysine (10% by volume) and lysine (20% by volume). 2 SO 4 Dry over medium, concentrate, and subject the resulting residue to column chromatography using CH 2 Cl 2 2%-5% MeOH in 4% was used as the eluent to obtain compound dUTP-FL-2 (348 mg, 63%) in the form of a foamy white solid. f 0.45(19:1DCM–MeOH) 1 HNMR(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).

[0445]

[0446] Synthesis of dUTP-FL-3: To a stirred solution of 2 (108 mg; 0.23 mmol) in DMSO (8 ml) were added acetic acid (4 ml) and acetic anhydride (12 ml). The reaction mixture was stirred at room temperature for 48 h. At 0 °C, saturated NaHCO 3 The solution (50 ml) was stirred for 30 min, and the aqueous layer was extracted with EtOAc (3×100 ml). The combined organic extracts were purified by Na 2 SO 4Dried and concentrated. The crude product was purified by flash column chromatography (ethyl acetate / hexane, 1:1 to 7:3) to afford dUTP-FL-3 (106 mg, 87%) as a foamy white solid. f 0.85 (1:1 hexane-EtOAc) 1 HNMR(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=56.2,1 1.5,2.3Hz),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).

[0447]

[0448] 6.12. Example 12. Design of the Synthesis of (L) 3'-O-Azidomethyl-7-deaza-dNTP-Label

[0449] 6.12.1.1 Scheme 19.NH 2 -L-3'-ON 3 Synthesis of -dCTP

[0450]

[0451] 6.12.1.2 Scheme 20.NH 2 -L-3'-ON 3 -dATP synthesis

[0452]

[0453] 6.12.1.3 Scheme 21.NH 2 -L-3'-ON 3 Synthesis of -dGTP

[0454]

[0455] 6.12.1.4 Scheme 23.NH 2 -(L)3'-ON 3 -Synthesis of 7-deaza-dATP

[0456]

[0457] NH2 -(L)3'-ON 3 -Synthesis scheme of 7-deaza-dATP:

[0458] At room temperature, 4-chloro-5-iodo-7H-pyrrolo[2.3-d]pyrimidine (1.0 g, 3.58 mmol) was added to CH 3 Powdered KOH (85%, 0.5 g, 7.57 mmol) and TDA-1 (0.075 mL, 0.24 mmol) were added to a solution in CH (60 mL). 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 filtrate was evaporated to dryness. The residue was applied to flash chromatography (silica gel, column 5 x 15 cm, eluted with petroleum ether-EtOAc, 4: 1). The combined fractions containing the product were evaporated to obtain 4-chloro-7-[2-deoxy-3,5-di-O-(4-methylbenzoyl-β-L-erythro-pentofuranosyl)-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (2.02 g, 89%) in the form of a colorless solid. The intermediate (1.8 g, 2.85 mmol) was dissolved in NH 3 / CH 3 A solution in OH (saturated at 0° C., 145 mL) was stirred at room temperature for 24 hours and the solvent was evaporated. Flash chromatography (silica gel, column 4×16 cm, CH 2 Cl 2 / CH 3 OH, 9:1) to give compound 7-deaza-dA-1 (0.45 g, 40%) as a colorless solid. f 0.45(CH 2 Cl 2 / CH 3 OH, 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 (CH 2 Cl 2 –CH 3 OH, 20:1) to afford 7-deaza-dA-2 (1.18 g, 89%) as a white solid. To a stirred solution of compound 7-deaza-dA-2 in DMSO were added acetic acid and acetic anhydride. The reaction mixture was stirred at room temperature for 48 h. Saturated NaHCO 3solution and the aqueous layer was washed with CH 2 Cl 2 The combined organic extracts were washed with saturated NaHCO 3 The solution was washed and then 2 SO 4 After concentration, the residue was purified by flash column chromatography (hexane / ethyl acetate, 1:1 to 1:4) to give compound 7-deaza-dA-3. Under nitrogen, compound 7-deaza-dA-3 was added in dry CH 2 Cl 2 Cyclohexene and SO 2 Cl 2 The reaction mixture was stirred at 0°C for 2 h. The solvent was removed first under reduced pressure and then under a high vacuum pump for 10 min. The residue was dissolved in dry DMF and mixed with NaN at room temperature. 3 The reaction was continued for 3 h. The reaction mixture was dispersed in distilled water (50 ml) and washed with CH 2 Cl 2 (3×50 ml) extraction. The combined organic layers were purified by Na 2 SO 4 The residue was dissolved in MeOH and mixed with NH 4 F was stirred for 24 h. The solvent was removed under reduced pressure. The reaction mixture was concentrated under reduced pressure and 2 O and CH 2 Cl 2 The organic layer was separated and purified by Na 2 SO 4 Dry. After concentration, the crude product was 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 solution of ammonia in methanol. The solution was stirred at 115°C-120°C for 15h. After evaporation, the residue was purified by flash column chromatography (CH 2 Cl 2 –CH 3 OH, 20:1) to give 7-deaza-dA-5. Tetrakis(triphenylphosphine)palladium(0) and CuI were added to a solution of 7-deaza-dA-5 in anhydrous DMF. The reaction mixture was stirred at room temperature for 10 minutes. N-propargyl trifluoroacetamide and Et 3 N was added to the above reaction mixture. The reaction was stirred at room temperature in the absence of air and light for 1.5 h. After evaporation, the residue was dissolved in ethyl acetate. The mixture was washed with saturated NaHCO 3 , NaCl aqueous solution and washed with anhydrous Na 2SO 4 After evaporation, the residue was purified by flash column chromatography (CH 2 Cl 2 –CH 3 OH, 10:1) to obtain 7-deaza-dA-6. Compound 7-deaza-dA-6 and proton sponge were dried in a vacuum dryer by P 2 O 5 Dry overnight and then dissolve in trimethyl phosphate. Freshly distilled POCl was then added dropwise at 0 °C. 3 , and the mixture was stirred at 0°C for 2 h. Subsequently, a well-swirled mixture of tributylammonium pyrophosphate and tributylamine in anhydrous DMF was added all at once and stirred for 30 min at room temperature. 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 NH 4 OH and stirred at room temperature overnight. The resulting mixture was concentrated under vacuum and the residue was diluted with 5 ml of water. The crude mixture was 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 was further purified by reverse phase HPLC to give (L) 3'-O-azidomethyl-7-deaza-dATP (compound 7-deaza-dA-7).

[0459] 6.12.1.5 Scheme 25. (L) 3'-ON 3 -Synthesis of 7-deaza-dATP-ROX

[0460]

[0461] Azide-ROX compounds (ROX- 3 -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 Application US20060160081A1) was dissolved in DMF (300 μl) and 1 M NaHCO 3 A solution of ROX NHS (N-hydroxysuccinimide) ester (Invitrogen) (0.013 mmol) in DMF (400 μl) was slowly added to the above reaction mixture, and then stirred for 5 h at room temperature in the absence of light. The crude product was purified on a preparative silica TLC plate (CHCl 3 / CH3 OH, 1:4).

[0462] L-3'-O-azidomethyl-7-deaza-dATP-ROX compound (L-3'-ON 3 -7-deaza-dATP-ROX). 3 -Linker was added to a stirred solution of DSC (N,N'-disuccinimidyl carbonate) (3.4 mg, 13.2 μmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 μmol) in dry DMF (2 ml). The reaction mixture was stirred at room temperature for 2 h. TLC showed that ROX-N 3 -The linker is completely converted to the compound ROX-N 3 - linker NHS ester, which is used directly in the reaction with NaHCO 3 / Na 2 CO 3 The reaction mixture was stirred at room temperature for 3 h in the absence of light. The reaction mixture was purified by preparative silica TLC plate (CH 3 OH / CH 2 Cl 2 The crude product was further purified on reverse phase HPLC to obtain L-3'-O-azidomethyl-dATP-ROX (L-3'-ON 3 -dATP-ROX).

[0463] 6.12.1.6 Scheme 26.NH 2 -(L)3'-ON 3 -Synthesis of denitrogenated dGTP

[0464]

[0465] NH 2 -(L)3'-ON 3 -Synthesis scheme of 7-deaza-dGTP. At room temperature, powdered KOH (85%, 1.15 g, 17.42 mmol) and TDA-1 (0.2 mL, 0.63 mmol) were added to CH 3 2-amino-4-chloro-7H-pyrrolo[2.3-d]pyrimidine (842 mg, 4.99 mmol) was added to the suspension in CN (60 mL). After stirring for 5 min, halogenated sugar (2.53 g, 6.51 mmol) was introduced within 15 min, and stirring was continued for 30 min. The insoluble material was filtered off and washed with CH 3The residue was subjected to flash chromatography (silica gel, column 6 x 12 cm, with CH 2 Cl 2 eluted). The combined fractions containing the product were evaporated to give a colorless solid (2.21 g, 85%). The intermediate (1.04 g, 2.00 mmol) was dissolved in 0.5 M NaOCH 3 / CH 3 The solution in 1% ethyl acetate (60 mL) was stirred at reflux for 3 h. The mixture was neutralized with AcOH and evaporated. The residue was applied to flash chromatography (silica gel, CH 2 Cl 2 / CH 3 OH, 95:5) to obtain compound 7-deaza-dG-1 (400 mg, 71%) in the form of a colorless solid. The stirred solution of 7-deoxy-dG-1 was co-evaporated with anhydrous pyridine (3×4 mL) and then dissolved in anhydrous pyridine (2 mL). The resulting solution was 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.) was added dropwise via a syringe. The ice bath was then removed and the mixture was stirred for 2 hours. The solution was cooled on ice, and isobutyryl chloride (0.29 mL, 15.69 mmol, 2.2 equiv.) was added dropwise via a syringe and the ice bath was removed. After stirring for another 2 hours at room temperature, the reaction was again placed on ice, and ice-cold water (20 mL) was slowly added, followed by concentrated ammonia solution (1.5 mL) after 15 minutes to obtain a final 2.5 M concentration of ammonia. The mixture was kept on ice for 30 minutes and then evaporated to dryness. The residue was co-evaporated with toluene (3 × 5 mL) to remove traces of water, resuspended in MeOH and filtered to remove precipitate. The filtrate was then concentrated, dissolved in a small amount of MeOH, adsorbed on silica gel and purified by column chromatography (DCM / MeOH 95:5 to 91:9 (v / v)) to obtain compound 7-denitrogenation-dG-2. Compound 7-denitrogenation-dG-2 was co-evaporated with dry pyridine three times, dried under high vacuum, and dissolved in 2 cm 3 in an ice bath. 3Dry N,N-dimethylformamide. Imidazole (419 mg, 6.15 mmol) was added, and the mixture was stirred at 0 ° C for 15 min and at room temperature for 15 min. Then, 241 mg of tert-butyldimethylsilyl chloride (1.59 mmol) was added, and the solution was stirred at 60 ° C for another 2 h. The mixture was diluted with dichloromethane, washed with brine, dried over sodium sulfate, and evaporated. The crude product was purified by column chromatography on silica gel (methanol: dichloromethane 0: 100–2: 98) as a white foam (compound 7-deaza-dG-3). NIS was added to a vigorously stirred solution of 7-deaza-dG-3 in anhydrous DMF. The reaction mixture was stirred at room temperature for 22 h, and most of the solvent was then removed under vacuum. Diethyl ether and saturated NaHCO were added. 3 The organic layer was washed with saturated NaCl and 2 SO 4 After evaporation, the residue was purified by flash column chromatography to give 7-deaza-dG-4. To a stirred solution of compound 7-deaza-dG-4 in DMSO (12 ml) were added acetic acid (5.5 ml) and acetic anhydride (17.6 ml). The reaction mixture was stirred at room temperature for 48 h. Saturated NaHCO 3 solution (100 ml) and the aqueous layer was washed with CH 2 Cl 2 The combined organic extracts were washed with saturated NaHCO 3 solution (100 ml) and washed with Na 2 SO 4 After concentration, the residue was purified by flash column chromatography to give compound 7-deaza-dG-5. Under nitrogen, compound 7-deaza-dG-5 was added in dry CH 2 Cl 2 Cyclohexene and SO 2 Cl 2 The reaction mixture was stirred at 0°C for 2 h. The solvent was removed first under reduced pressure and then under a high vacuum pump for 10 min. The residue was dissolved in dry DMF and mixed with NaN at room temperature. 3 The reaction was continued for 3 h. The reaction mixture was dispersed in distilled water (50 ml) and washed with CH 2 Cl 2 (3×50 ml) extraction. The combined organic layers were purified by Na 2 SO 4The residue was purified by flash column chromatography (ethyl acetate / methanol, 100:0 to 98:2) to give compound 7-deaza-dG-6. Tetrakis(triphenylphosphine)palladium(0) and CuI were added to a solution of 7-deaza-dG-6 in anhydrous DMF. The reaction mixture was stirred at room temperature for 10 minutes. N-propargyl trifluoroacetamide and Et 3 N was added to the above reaction mixture. The reaction was stirred at room temperature in the absence of air and light for 1.5 h. After evaporation, the residue was dissolved in ethyl acetate. The mixture was washed with saturated NaHCO 3 , NaCl aqueous solution and washed with anhydrous Na 2 SO 4 After evaporation, the residue was purified by flash column chromatography to give 7-deaza-dG-7. 7-deaza-dG-7 was reacted with 4% paraformaldehyde in anhydrous CHCl. 3 NaI and chlorotrimethylsilane were added to the stirred solution in CN. 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. A solution of tetrabutylammonium fluoride (TBAF) in THF 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 NaCl aqueous solution and washed with anhydrous Na 2 SO 4 After evaporation of the solvent, the residue was purified by flash column chromatography (EtOAc–CH 3 OH) to obtain 7-deaza-dG-8. Compound 7-deaza-dG-8 and proton sponge were separated in a vacuum desiccator by P 2 O 5 Dry overnight and then dissolve in trimethyl phosphate. Freshly distilled POCl was then added dropwise at 0 °C. 3 , and the mixture was stirred at 0°C for 2 h. Subsequently, a well-swirled mixture of tributylammonium pyrophosphate and tributylamine in anhydrous DMF was added all at once and stirred for 30 min at room temperature. 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 NH 4 OH and stirred at room temperature overnight. The resulting mixture was concentrated under vacuum and the residue was diluted with 5 ml of water. The crude mixture was 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 was further purified by reverse phase HPLC to obtain (L) 3'-O-azidomethyl-7-deaza-dATP (compound 7-deaza-dG-9).

[0466] 6.12.1.7 Scheme 27. (L) 3'-ON 3 -7-deaza-dGTP-Cy5

[0467]

[0468] Azide-Cy5 compounds (Cy5-N 3 -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 Application US20060160081A1) was dissolved in DMF (300 μl) and 1 M NaHCO 3 A solution of Cy5 NHS (N-hydroxysuccinimide) ester (Invitrogen) (0.013 mmol) in DMF (400 μl) was slowly added to the above reaction mixture, and then stirred for 5 h at room temperature in the absence of light. The crude product was purified by preparative silica gel TLC plate (CHCl 3 / CH 3 OH, 1:4).

[0469] L-3'-O-azidomethyl-7-deaza-dGTP-Cy5 compound (L-3'-ON 3 -7-deaza-dGTP-Cy5). 3 -Linker was added to a stirred solution of DSC (N,N'-disuccinimidyl carbonate) (3.4 mg, 13.2 μmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 μmol) in dry DMF (2 ml). The reaction mixture was stirred at room temperature for 2 h. TLC showed that Cy5-N 3 -The linker is completely converted into the compound Cy5-N 3 - linker NHS ester, which is used directly in the reaction with NaHCO 3 / Na 2 CO 3 The reaction mixture was stirred at room temperature for 3 h in the absence of light. The reaction mixture was then separated by preparative silica gel TLC plates (CH 3 OH / CH 2 Cl 2The crude product was further purified on reverse phase HPLC to obtain L-3'-O-azidomethyl-dGTP-Cy5 (L-3'-ON 3 -dGTP-Cy5).

[0470] 6.13. Example 13. Chemical synthesis of (D)-type 9°N DNA polymerase via solid phase peptide synthesis and native chemical ligation

[0471] Figure 1 The amino acid sequence of the (D)-type mutant 9°N DNA polymerase (candidate #1-1) is provided, in which all amino acids are D-type amino acids. Diamonds = mutations introduced for NCL; circles = NCL site (start of the 'second' chain); squares = potential substitution sites for isoleucine; triangles = mutations introduced for modified nucleotides.

[0472]

[0473] 9°N DNA polymerase was allowed to cleave into two fragments (466-aa 9°NN fragment and 310-aa 9°NC fragment) at the cleavage 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 ( Figure 2A ), which are linked at certain cysteine ​​residues, such as Figure 2B Similarly, the 310-aa 9° NC fragment was split into six synthetic peptides ( Figure 3A ), which are synthesized via solid phase peptide synthesis and then linked at certain cysteine ​​residues, such as Figure 3B as shown in .

[0474] 6.13.1. Experimental methods:

[0475] Materials: 2-Chlorotrityl chloride resin (load = 0.98 mmol g -1 ) was purchased from Purepep. Fmoc-D-amino acids, D-4-thiazolidinecarboxylic 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 used in peptide synthesis were purchased from Chempep, Sigma-aldrich, Alfa aesar, TCI, etc. Reagents used in NCL reactions, i.e., guanidine hydrochloride (Gn·HCl), Na 2 HPO 4 12H 2 O、NaH2 PO 4 ·2H 2 O, sodium nitrite (NaNO 2 ), 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 (PdCl 2 ) were purchased from Sigma-aldrich, Alfaaesar, TCI, Duksan, etc.

[0476] Fmoc-based SPPS. All peptides were synthesized by Fmoc-based SPPS on a Liberty Blue automated microwave peptide synthesizer (CEM) and Synthesized on a 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. Prior to coupling, all resins were swollen in DMF for 30 min. The Fmoc group of the assembled amino acids was removed by treatment with 20% piperidine and 0.1 M Oxyma in DMF at 85°C. The coupling of amino acids other than Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH was performed at 85°C using 5 equivalents of amino acid, 5 equivalents of Oxymapure, and 10 equivalents of DIC for 2 min. The coupling reaction for Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH was performed at 50°C for 10 min to avoid side reactions at high temperatures. Trifluoroacetylthiazolidine-4-carboxylic acid-OH was coupled with 5 equivalents of Oxymapure and 10 equivalents of DIC at room temperature overnight. For peptides longer than 20 amino acids, a double coupling strategy was used. After the peptide chain was assembled, H 2 The peptide was cleaved from the resin using 1% ethyl acetate (0.5 / 0.5 / 0.5 / 0.25 / 8.25) (vol / vol). The cleavage reaction was carried out at 27° C. with stirring for 2.5 h. 1% 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 CH 3 CN / H 2 O, analyzed by RP-HPLC and purified by semi-preparative HPLC. The collected peptide fractions were analyzed by electrospray ionization mass spectrometry (ESI-MS).

[0477] Native Chemical Ligation (NCL). The C-terminal peptide hydrazide segment was dissolved in acidified ligation buffer (6 M Gn·HCl and 0.1 M NaH 2 PO 4 The mixture was cooled in an ice-salt bath (-15°C) and 10 equivalents of NaNO in acidified ligation buffer (pH 3.0) were added. 2 . The activated reaction system was kept in an ice-salt bath for 20 min under stirring, after which 40 equivalents of MPAA and 1 equivalent of N-terminal cysteine ​​peptide in the connection buffer were added, and the pH of the solution was adjusted to 6.6-6.8 at room temperature. After the overnight reaction, 150 mM TCEP in the connection buffer (pH adjusted to 7.0) was added to dilute the system twice, and the reaction system was kept at room temperature for 30 min under stirring. Finally, the connection product was analyzed by HPLC and purified by semi-preparative HPLC. The purified connection fraction was analyzed by ESI-MS.

[0478] Synthesis of Tfa-D-Thz-OH:

[0479]

[0480] To a solution of D-4-thiazolidinecarboxylic acid (1 g, 7.509 mmol) suspended in MeOH (40 mL) was added triethylamine (2.62 mL, 18.772 mmol), 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 (0.9 g, 3.92 mmol, 52.3%) ( Figure 4A ). 1 H NMR (500 MHz, CDCl 3 ):δ(ppm)9.35(s,1H),5.09(dd,J=6.9,4.2Hz,1H),4.86-4.77(m,1H,rotamer),4.75-4.61(m,1H,rotamer),3.52-3.28(m,2H,rotamer)( Figure 4B ).

[0481] Fmoc-based SPPS ( Figure 5 ):

[0482] 1) 102 mg of 2-Cl-(Trt)-Cl resin (0.98 mmol g -1 ) into the peptide synthesis vessel.

[0483] 2) Add 5 ml of DMF to the resin from the top of the reaction vessel, stir it gently for 10 s, and then drain it.

[0484] 3) Repeat step 2 two more times.

[0485] 4) Add 5 ml of DCM to the resin, stir it gently for 10 s, and then drain it.

[0486] 5) Repeat step 4 two more times.

[0487] 6) Repeat step 2 three more times.

[0488] 7) The resin was swelled in 4 ml of 50% (v / v) DMF / DCM for 30 min and then drained.

[0489] 8) Add 4 ml of 5% (vol / vol) NH 2 NH 2 .H 2 O was used for hydrazine reaction. The mixture was gently stirred at 30°C in a thermostatic oscillator for 30 min, and then the solution was discharged by vacuum filtration.

[0490] 9) Add 4 ml of DMF to the resin, stir it gently for 10 s, and then drain it.

[0491] 10) Repeat step 8.

[0492] 11) Wash the resin by repeating steps 2-6 twice.

[0493] 12) Add 4 ml of 5% (v / v) MeOH / DMF to the resin, stir it gently for 10 min, and then drain it to the unreacted sites on the resin to be primed.

[0494] 13) Repeat steps 2-6 to fully wash the resin.

[0495] 14) Add 4 ml of DCM to the resin, stir it gently for 10 s, and then drain it off. Use the resin directly for the next coupling step.

[0496] 15) For storage, the resin was dried under high vacuum for 2 h and the dried 2-Cl-(Trt)-NHNH 2 The resin was stored at -20 °C under argon for up to 2 weeks.

[0497] Solid-phase peptide synthesis of D-9°NN-6@5-mer: Tfa-Thz-AVYE-NHNH 2 ( Fig. 6A ). By following the conditions mentioned in the experimental method, D-9°NN-6@5-mer was synthesized on a Chorus automated peptide synthesizer. The D-9°NN-6@5-mer synthesized by this method was analyzed by HPLC chromatogram and the results are provided in Figure 6B-6C middle.

[0498] Solid-phase peptide synthesis of D-9°NN-6@11-mer: CVFGKPKEKVY-NHNH 2 :By following the conditions mentioned in the experimental method, D-9°NN-6@11-mer was synthesized on a Chorus automated peptide synthesizer. The D-9°NN-6@11-mer synthesized by this method was analyzed by HPLC chromatogram and the results are provided in Figures 7B-7D middle.

[0499] Solid-phase peptide synthesis of D-9°NN-6@24-mer: CEEIAQAWE SGEGLERVAR YSMED-NHNH 2 (SEQ ID NO:13) Fig. 8A ). By following the conditions mentioned in the experimental method, D-9°NN-6@24-mer was synthesized on a Chorus automated peptide synthesizer. The D-9°NN-6@24-mer synthesized by this method was analyzed by HPLC chromatogram and the results are provided in Figures 8B-8C middle.

[0500] Solid-phase peptide synthesis of D-9°NC-3@9-mer: CDTDGLHAT-NHNH 2 (SEQ ID NO:14) Fig. 9A ). D-9°NC-3@9-mer was synthesized on a Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) by following the conditions mentioned in the experimental method. D-9°NC-3@9-mer synthesized by this method was analyzed by HPLC chromatogram and the results are provided in Figure 9B-9C middle.

[0501] Custom solid phase peptide synthesis of D-9°NC-1@33-mer: MKATVDPLEK KLLDYRQRLI KILANSFYGYYGY-NHNH2. D-9°NC-1@33-mer synthesized by this method was analyzed by HPLC chromatogram and the results are provided at Figures 10A-10B middle.

[0502] Custom solid phase peptide synthesis of D-9°NC-2@39-mer: CKARWY-C(Acm)-KE-C(Acm)AESVTAWGREYIEMVIRELE EKFGFKVLY-NHNH 2 The D-9°NC-2@39-mer synthesized by this method was analyzed by HPLC chromatogram and the results are provided in Figures 11A-11B middle.

[0503] Solid phase peptide synthesis of D-9°NC-3@21-mer: TFA-Thz-DTDGLHATIPGADAETVKKK-NHNH2 (SEQ ID NO: 15). D-9oN-C-3@21-mer was synthesized on a Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) by following the conditions mentioned in the experimental method. The product was observed to be TFA-deprotected D-9°NC-3@21-mer, as shown in FIG. Fig. 12B and 12C provided in.

[0504] Solid phase peptide synthesis of D-9°NC-3@Cys35-mer: CKEFLKYINP KLPGLLELEY EGFYVRGFFVTKKKY-NHNH 2 (SEQ ID NO: 16). D-9°NC-3@Cys35-mer was synthesized on a Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) by following the conditions mentioned in the experimental methods. The product was observed to be of the expected quality, as Figures 13B-13D provided in.

[0505] Solid phase peptide synthesis of D-9°NC-3@56-mer: CDTDGLHATIPGADAETVKKKAKEFLKYINPKLPGLLELEYEGFYVRGFF VTKKKY-NHNH 2 (SEQ ID NO:17) Fig.14A ). D-9°NC-3@56-mer was synthesized on a Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) by following the conditions mentioned in the experimental method. The product was observed to be of the expected quality, as Fig. 14B provided in.

[0506] D-9°NC-3@35-mer:AKEFLKYINP KLPGLLELEY EGFYVRGFFV TKKKY-NHNH 2 (SEQ ID NO:18) Fig.15A ). D-9°NC-3@35-mer was synthesized on a Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) by following the conditions mentioned in the experimental method. The product was observed to have the expected quality, as Fig. 15B provided in.

[0507] Native Chemical Ligation (NCL): Reagent preparation: NaOH, 1M and 6M. Dissolve 40 mg or 240 mg of NaOH in 1 ml of deionized H 2 HCl, 6M. Mix 1 ml of 12M HCl with 1 ml of deionized H 2 O. Two different phosphate solutions (0.1 M) containing 6 M Gn·HCl (pH 3.0–3.1 and pH 5.7–6.0, respectively). For a 10-ml solution, 156 mg of NaH 2 PO 4 ·2H 2 O and 5.74 g of Gn·HCl were mixed into a 10-ml volumetric flask and washed with deionized H 2 O to the final volume. The pH was adjusted to 3.0–3.1 with 6 M NaOH and 6 M HCl. The solution was filtered by using a 13 mm × 0.22 μm microporous membrane filter. The filtered solution can be stored at 4 °C for at least 1 month. 2 HPO 4 12H 2 O (0.1 M) (pH 5.7-6.0) was also prepared in the same way. 2 , 0.5M. 17 mg of NaNO 2 Dissolve in 0.5 ml of deionized HO 2O. TCEP, 0.1 M. 58 mg of TCEP·HCl was dissolved in 1 ml of 0.2 M phosphate solution (pH 3.0) containing 6 M guanidine hydrochloride (Gn·HCl). The pH was adjusted to 6.0–7.0, and the solution was filtered using a 13 mm×0.22 μm microporous membrane filter. VA-044, 0.1 M. 9.7 mg of VA-044 was weighed into a 2-ml Eppendorf reaction tube, and 0.3 ml of 0.2 M phosphate solution (pH 6.9–7.0) containing 6 M Gn·HCl was added. VA-044 was completely dissolved by using a vortex and ultrasonic cleaning bath.

[0508] Synthesis of D-9°NN-6@16-mer: Tfa-Thz-AVYECVFGKPKEKVY-NHNH 2 (SEQ ID NO: 19 and SEQ ID NO: 20) ( Fig.16A ). Fig. 16B The method for preparing D-9°NN-6@16-mer is shown in , and the HPLC chromatogram analysis of D-9°NN-6@16-mer is provided in Fig. 16C middle.

[0509] Synthesis of D-9oN-C-7@72-mer: MKATVDPLEKKLLDYRQRLIKILANSFYGYYGYCKARWY-C(Acm)-KE-C(Acm)AESVTAWGRE YIEMVIRELE EKFGFKVLY-NHNH2(SEQ ID NO:21, 22, 23)( Fig.17A ). Fig. 17B The method for preparing D-9oN-C-7@72-mer is shown in FIG. Fig. 17C is an analytical HPLC chromatogram (λ=214 nm) of the peak transitions during the NCL reaction progress. Fig.17D is the ESI-MS of D-9°NC-7@72-mer with MPAA attached, and Fig.17E A connection with MPAA attached is shown.

[0510] 7. Equivalents and Incorporation by Reference

[0511] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

[0512] All references, issued patents, and patent applications cited in the text of this specification are hereby incorporated by reference in their entirety for all purposes.

[0513] 8. Sequence

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

Claims

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

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

3. The nucleotide of claim 1, having a structure according to formula I: wherein the base is a nitrogenous base and R' is a cleavable protecting group.

4. The nucleotide of claim 1 having a structure according to formula II wherein the base is a nitrogenous base; R' is a cleavable protecting group or H; and R2-label is a cleavable label comprising a cleavable linker R2 and a label.

5. The nucleotide of claim 1, having a structure according to Formula III: The base is a nitrogenous base; and R2-label is a cleavable label comprising a cleavable linker R2 and a label.

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

7. The nucleotide of any one of claims 1-4, wherein the cleavable protecting group is selected from the group consisting of allyl, dimethyldisulfide, nitrobenzyl and azido protecting groups.

8. The nucleotide of claim 7, wherein the cleavable protecting group is selected from:

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

11. The nucleotide of claim [0124], wherein the cleavable linker comprises:

12. The nucleotide of claim 3, selected from the group consisting of: Wherein said R' is selected from:

13. The nucleotide of claim 4, selected from the group consisting of: Wherein said R' is selected from: R2 contains:

14. The nucleotide of claim 5, selected from the group consisting of: Where R2 contains:

15. The nucleotide of claim 4, selected from the group consisting of: Wherein said R' is selected from:

16. The nucleotide of claim 5, selected from the group consisting of: Wherein said R' is selected from:

17. A mirror 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.

18. The polymerase of claim 17, wherein the polymerase consists of D-amino acids.

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

1.

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. The polymerase of any one of claims 17-20, comprising one or more modifications compared to SEQ ID NO: 1 at one or more amino acid positions selected from E276, K317, N424 and S651.

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

1.

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

1.

24. The polymerase of any one of claims 17-23, comprising one or more modifications compared to SEQ ID NO: 1 at one or more amino acid positions selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733, and I744.

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

1.

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. The polymerase of claim 26, comprising substitutions of 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.

28. The polymerase of any one of claims 17-27, comprising one or more modifications compared to SEQ ID NO: 1 at one or more amino acid positions selected from the group consisting of M129, 1130, G131, D141, E143, L408, Y409, P410, A485, T514, and 1521.

29. The polymerase of claim 28, comprising one or more modifications compared to SEQ ID NO: 1 at one or more amino acid positions selected from the group consisting of D141, E143, Y409 and A485.

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

1.

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

1.

32. The polymerase of claim 28, comprising one or more modifications compared to SEQ ID NO: 1 at one or more amino acid positions selected from the group consisting of D141, E143, L408, Y409, P410, A485, T514, and I521.

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

1.

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

1.

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

1.

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

1.

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

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

39. A method for replicating an L-polynucleotide, comprising the steps of: A mixture comprising (i) the L-polynucleotide, (ii) an L-primer, (iii) L-dNTPs, (iv) the polymerase of any one of claims 1 to 38, and (v) a buffer is incubated to induce replication of the L-polymerase.

40. The method of claim 39, wherein the L-polynucleotide is DNA or RNA.

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

42. The method of any one of claims 39-41, wherein the buffer comprises 50 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 1 mM DTT, and 50 mM KCl.

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

44. A method for sequencing an L-polynucleotide, comprising the following cycles: a. incubating a mixture comprising (i) the 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, thereby obtaining a replication product; b. detecting a signal from the L-3'-O-R'-dNTP-R2-label incorporated into the replication product; and c. Inducing cleavage of the L-3'-O-R'-dNTP-R2-labeled R' group and R2 group incorporated into the replication product.

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 method of any one of claims 44-45, wherein the L-3'-O-R'-dNTP-R2-label comprises a L-3'-O-R'-dATP-R2-label, a L-3'-O-R'-dTTP-R2-label, a L-3'-O-R'-dGTP-R2-label, and a L-3'-O-R'-dCTP-R2-label, wherein each label is different.

47. The method of any one of claims 44-46, wherein the L-3'-O-R'-dNTP-R2-label has a structure according to Formula II as defined in any one of claims 4, 6-13 and 15.

48. The method of claim 47, wherein the L-3'-O-R'-dNTP-R2-label is the nucleotide of claim 13 or 15.

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

50. A method for sequencing an L-polynucleotide, comprising the steps of: a. incubating a mixture comprising (i) the L-polynucleotide, (ii) an L-primer, (iii) L-dNTP, (iv) an L-ddNTP-R2-label or an L-3'-O-R'-dNTP-R2-label, (v) a polymerase according to any one of claims 17 to 38, and (vi) a buffer, thereby obtaining a replication product; b. separating the replication product; and c. Detecting the signal from the L-ddNTP-R2-label or L-3'-O-R'-dNTP-R2-label incorporated into the replication product.

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

52. The method of claim 50 or 51, wherein 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, wherein each label is different.

53. The method of any one of claims 50-52, wherein the L-ddNTP-R2-label has formula III as defined in any one of claims 5, 6, 9-11.

54. The method of claim 53, wherein the L-ddNTP-R2-label is a nucleotide of claim 14 or 16.

55. The method of claim 50 or 51, wherein the L-3'-O-R'-dNTP-R2-label comprises a L-3'-O-R'-dATP-R2-label, a L-3'-O-R'-dTTP-R2-label, a L-3'-O-R'-dGTP-R2-label, and a L-3'-O-R'-dCTP-R2-label, wherein each label is different.

56. The method of any one of claims 50-51 and 55, wherein the L-3'-O-R'-dNTP-R2-label has Formula II as defined in any one of claims 4, 6-13 and 15.

57. The method of claim 56, wherein the L-3'-O-R'-dNTP-R2-label is the nucleotide of claim 13 or 15.

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

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

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

61. A method for sequencing an L-polynucleotide, comprising the following cycles: a. incubating a mixture comprising (i) the L-polynucleotide, (ii) an L-primer, (iii) L-3'-O-R'-dNTP, (iv) an L-ddNTPs-R2-label or an L-dNTPs-R2-label, (v) a polymerase according to any one of claims 17 to 38, and (vi) a buffer, thereby obtaining a replication product; b. detecting a signal from the L-ddNTP-R2-label or L-3'-O-R'-dNTP-R2-label incorporated into the replication product; and c. Inducing i) the cleavage of the R' group of the L-3'-O-R'-dNTP and the R2 group of the L-ddNTPs-R2-labeled incorporated into the replication product; or ii) the 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-labeled incorporated into the replication product.

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 61-62, wherein the L-3'-O-R'-dNTPs comprise L-3'-O-R'-dATP, L-3'-O-R'-dTTP, L-3'-O-R'-dGTP, and L-3'-O-R'-dCTP.

64. The method of any one of claims 61-63, 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 the nucleotide of claim 12.

66. The method of any one of claims 61-65, wherein 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, wherein each label is different.

67. The method of any one of claims 61-66, wherein the L-ddNTP-R2-label has a structure according to Formula III as defined in any one of claims 5, 6, and 9-11.

68. The method of claim 67, wherein the L-ddNTP-R2-label is a nucleotide of claim 14 or 16.

69. The method of any one of claims 61-65, wherein the L-3'-O-R'-dNTP-R2-label comprises a L-3'-O-R'-dATP-R2-label, a L-3'-O-R'-dTTP-R2-label, a L-3'-O-R'-dGTP-R2-label, and a L-3'-O-R'-dCTP-R2-label, wherein each label is different.

70. The method of any one of claims 61-66 and 69, wherein the L-3'-O-R'-dNTP-R2-label has a structure according to Formula II as defined in any one of claims 4, 6-13 and 15.

71. The method of claim 67, wherein the L-3'-O-R'-dNTP-R2-label is the nucleotide of claim 13 or 15.

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

73. A kit for replicating an L-polynucleotide comprising (i) the polymerase of any one of claims 17-38 and (ii) optionally a buffer.

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. The kit of any one of claims 73-75, further comprising a L-3'-O-R'-dNTP-R2-label.

77. The kit of claim 76, wherein 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, an L-3'-O-R'-dCTP-R2-label or an L-3'-O-R'-dUTP-R2-label.

78. The kit of any one of claims 76-77, wherein the L-3'-O-R'-dNTP-R2-label has Formula II as defined in any one of claims 4-11.

79. The kit of claim 78, wherein the L-3'-O-R'-dNTP-R2-label is the nucleotide of claim 13 or 15.

80. The kit of any one of claims 73-79, further comprising a L-ddNTP-R2-label.

81. The kit of claim 80, wherein the L-ddNTP-R2-label comprises 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.

82. The kit of any one of claims 76-81, wherein the L-ddNTP-R2-label has Formula III as defined in any one of claims 5, 6, and 9-11.

83. The kit of claim 82, wherein the L-ddNTP-R2-label is a nucleotide of claim 14 or 16.

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

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

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

Images