Nucleotide analogue, kit and application

Abstract

The invention discloses a nucleotide analogue, a kit and application. The nucleotide analogue disclosed by the invention has a structure as shown in a formula I, wherein R1 is adenine, guanine, cytosine, thymine, uracil or analogues of the adenine or the pyrimidine; r2 / R3 is a fluorophore, a reactive active group, H or a molecule for increasing the binding capacity with a DNA chain; r4 is a monophosphate group or a polyphosphate group, and in the polyphosphate group, O between a first phosphate group and a second phosphate group at the 5'end is substituted by CH2; and L1 is a linker. The nucleotide analogue disclosed by the invention solves the problem that a chemical structure'scar 'is easy to remain in the elution process of a fluorescent dye connecting chain, and also solves the problems that the flexibility of a newly synthesized chain and the combination efficiency of polymerase are influenced, the base mismatch probability is increased, the sequencing read length and the data quality are limited and the like; longer read length sequencing can be realized, and the method has higher quality and lower error rate.

Description

Technical Field

[0001] This application relates to the field of gene sequencing technology, and in particular to a nucleotide analog, a reagent kit, and its application. Background Technology

[0002] With the rapid development of life sciences, the demand for DNA sequencing technology is becoming increasingly urgent. The completion of the complete human genome sequence has made research into identifying genetic links behind common diseases through SNP mapping or direct association a hot topic. To achieve partial sequencing of a large portion of the population to obtain SNP and SV profiles for specific diseases, rapid, high-throughput, and low-cost DNA sequencing technologies are needed. While traditional Sanger sequencing is widely used, it has limitations such as high cost and low throughput. Next-generation sequencing (NGS) technology has made progress, such as generating large amounts of data, but it still needs continuous improvement, such as reducing costs and increasing sequencing accuracy and length. Developing novel sequencing technologies, especially innovations in compounds and compositions, is of great significance for advancing multiple research fields, including comparative genomics, evolutionary biology, forensic medicine, epidemiology, and applied medicine.

[0003] Current high-throughput gene sequencing platforms generally use reversible termination nucleotides (NRTs) for SBS sequencing, which achieves base recognition by modifying nucleotides with fluorescent dyes and cleavable protecting groups. However, this technology has inherent drawbacks: the linker strands of fluorescent dyes are prone to leaving chemical structural "scars" during elution, affecting the flexibility of the newly synthesized strands and the binding efficiency of polymerase, leading to an increased probability of base mismatches and limiting sequencing read length and data quality.

[0004] Therefore, how to solve the technical problems such as base mismatch caused by the defects and deficiencies of existing reversible termination nucleotides remains a key research focus and challenge in the field of high-throughput sequencing technology. Summary of the Invention

[0005] The purpose of this application is to provide an improved nucleotide analog, a kit using the nucleotide analog, and the application of the nucleotide analog and kit of this application.

[0006] To achieve the above objectives, this application adopts the following technical solution:

[0007] The first aspect of this application discloses a nucleotide analog having the structure shown in Formula 1;

[0008] Formula 1 ;

[0009] In Formula 1, R1 is adenine (A), guanine (G), cytosine (C), thymine (T), uracil (U), or analogs of these purines or pyrimidines; R2 / R3 are fluorescent groups, reactive groups, H, or molecules that increase the binding ability to the DNA chain; R4 is a monophosphate group or a polyphosphate group, and in the polyphosphate group, the O between the first and second phosphate groups at the 5' end is replaced by CH2, NH, S, CF2, CBr2, or Se; L1 is a linker. The substitution of O with CH2 allows for simple synthesis based on the existing O, and the bond formed by PCP cannot be converted into a phosphodiester bond under the action of DNA polymerase to attach to the DNA chain.

[0010] It should be noted that the nucleotide analogs of this application can be recognized by polymerase and incorporated into the growing DNA strand. However, due to the design of R4, only an intermediate complex of "template-polymerase-nucleotide analog" can be formed. Base recognition is achieved through fluorescence signals. After imaging, there is no need for excision. The nucleotide analogs can be directly removed by elution with washing buffer or by competitively replacing them with dNTPs containing blocking groups to extend the DNA strand of the sequencing primer. After the dNTPs with blocking groups are extended, their blocking groups can be removed to prepare for the next round of sequencing. In other words, the sequencing method based on the nucleotide analogs of this application achieves scarless incorporation of natural bases through competitive substitution and orthogonal cleavage reactions. Therefore, sequencing using the nucleotide analogs of this application solves the problem of residual chemical structure "scars" in the linker strand of fluorescent dyes during elution, and also solves the problems caused by this, such as affecting the flexibility of the newly synthesized strand and the binding efficiency of polymerase, which in turn leads to an increased probability of base mismatch, limiting sequencing read length and data quality.

[0011] It should also be noted that the key to this application lies in the nucleotide analogs with the structure shown in Formula 1. As for the linkers, fluorescent groups, etc., they can be designed and selected with reference to existing sequencing methods, and are not specifically limited here.

[0012] In one implementation of this application, the nucleotide analogue includes at least one of compound 1, compound 2, compound 3, and compound 4;

[0013] Compound 1 ;

[0014] Compound 2 ;

[0015] Compound 3 ;

[0016] Compound 4 .

[0017] In compounds 1 to 4, L1 is a linker, and Dye1, Dye2, Dye3 and Dye4 are different fluorescent groups.

[0018] In one implementation of this application, the fluorescent groups of compounds 1 to 4 are non-repeatingly selected from AF488, AF532, AF633, AF680, AF660, AF700, AF647, AF594, AF555, AF568, CY3, CY5, CY5.5, CY7, CY7.5, ROX, R6G, ATTO495, ATTO532, ATTO700, ATTO680, ATTO655, ATTO647N, ATTO594, ATTORho101, ATTO590, ATTO Thio12, FAM, VIC, TET, JOE, HEX, CAL Fluor Orange560, TAMRA, CALFluor Red610, TEXAS RED, and CAL Fluor. Red635, iFluor488, iFluor514, iFluor532, iFluor546, iFluor555, iFluor568, iFluor590, i Fluor610, iFluor633, iFluor647, iFluor680, iFluor700, iFluor710, Quasar705, Quasar670.

[0019] In one implementation of this application, the connector is either a splittable connector or a non-splittable connector.

[0020] It should be noted that the linker is the molecular part that forms a covalent link between the nucleotide and the fluorescent label. Depending on the requirements, either a non-cleavable or cleavable linker can be selected.

[0021] In one implementation of this application, the cleavable linker includes at least one of the following: electrophilic cleavage linker group, nucleophilic cleavage linker group, photolytic linker group, cleavage group under reducing conditions, cleavage group under oxidizing conditions, safety handle type linker group, and group cleaved by elimination mechanism.

[0022] In one implementation of this application, the cleavable linker is at least one selected from alkyl, allyl, azidomethylene, 2-nitrobenzyl, and dithio.

[0023] In one implementation of this application, the non-cleavable linker includes at least one of a polyethylene glycol chain and a polypeptide chain. The polyethylene glycol chain can be formed by the polymerization of one or more ethylene glycols; similarly, the polypeptide chain can consist of one or more amino acids.

[0024] A second aspect of this application discloses a kit comprising the nucleotide analogue of this application.

[0025] In one implementation of this application, the kit further includes a reversible blocking nucleotide derivative having the structure shown in Formula 2.

[0026] Formula 2 ;

[0027] In Formula 2, R5 is a 3'-OH modifying group that can be orthogonally cleaved, i.e., a reversible blocking group; R6 is adenine A, guanine G, cytosine C, thymine T, or uracil U; R7 is a monophosphate group or polyphosphate group that can normally form a phosphodiester bond.

[0028] It should be noted that in the kit of this application, the nucleotide analogue used in this application serves as a "recognition probe," which can be recognized by polymerase and incorporated into the growing DNA strand. However, due to the design of R4, it does not participate in DNA strand elongation; it merely forms a "template-polymerase-nucleotide analogue" complex. Base recognition is achieved through fluorescence signals. After signal collection, it is either directly eluted with washing buffer or competitively replaced by a reversible blocking nucleotide derivative. The reversible blocking nucleotide derivative in the kit of this application serves as a base synthesis reagent. After base recognition, it competitively replaces the nucleotide analogue in the "template-polymerase-nucleotide analogue" complex, forming a stable phosphodiester bond through polymerase catalysis. The blocking group at the 3' end of the reversible blocking nucleotide derivative can be removed by cleavage solution, generating a natural 3'-OH, preparing for the next round of sequencing.

[0029] In one implementation of this application, the reversible blocking group of the reversible blocking nucleotide derivative of this application includes at least one of methylene azide, allyl, ester, phosphoric acid, hydroxylamine, disulfide bond, photolytic group, 2-nitrobenzyl, and azo compound.

[0030] In one implementation of this application, the reversible blocking group includes at least one of the following groups.

[0031] .

[0032] In one implementation of this application, the reversible blocking nucleotide derivative includes at least one of compound 5, compound 6, compound 7, and compound 8;

[0033] Compound 5 ;

[0034] Compound 6 ;

[0035] Compound 7 ;

[0036] Compound 8 ;

[0037] In compounds 5 to 8, N3 is the methylene azido blocking group.

[0038] In one implementation of this application, the kit further includes at least one of polymerase, cleavage buffer, and scanning buffer; the cleavage buffer contains at least one cleavage agent selected from Na2S2O4, THPP, TECP, and DTT, for specifically removing the blocking group of the reversible blocking nucleotide derivative; the scanning buffer is used to adjust the pH and polarity of the reaction environment to suit the needs of fluorescence excitation or quenching.

[0039] It should be noted that the key to the kit in this application lies in the use of nucleotide analogs with the structure shown in Formula 1 and reversible blocking nucleotide derivatives with the structure shown in Formula 2. As for other reagents, such as polymerase, cutting buffer and scanning buffer, existing sequencing methods can be referenced.

[0040] The third aspect of this application discloses the use of the nucleotide analogues or kits of this application in nucleic acid sequencing.

[0041] Due to the adoption of the above technical solutions, the beneficial effects of this application are as follows:

[0042] The nucleotide analogues of this application solve the problem of residual chemical structure "scars" in the linker strand of fluorescent dyes during elution, and also solve the problems that lead to the reduced flexibility of the newly synthesized strand and the binding efficiency of polymerase, resulting in an increased probability of base mismatch, limiting sequencing read length and data quality. It enables sequencing with longer read lengths, higher quality and lower error rate, and provides a new reagent for high-throughput sequencing. Detailed Implementation

[0043] Current mainstream next-generation sequencing platforms still suffer from short read lengths and error rates mostly exceeding 0.1%. This application addresses this issue by synthesizing fluorescently modified dNTPs to avoid scarring after excision, and employing an optimized sequencing protocol system to achieve longer read lengths, higher quality, and lower error rates.

[0044] The nucleotide analogues of this application have the structure shown in Formula 1;

[0045] Formula 1 ;

[0046] In Formula 1, R1 is adenine A, guanine G, cytosine C, thymine T, uracil U, or analogs of these purines or pyrimidines; R2 / R3 is a fluorescent group, a reactive group, H, or a molecule that increases the ability to bind to the DNA strand; R4 is a monophosphate group or a polyphosphate group, wherein the O between the first phosphate group and the second phosphate group at the 5' end of the polyphosphate group is replaced by CH2; L1 is a linker.

[0047] In use, the four nucleotide analogs, R1, are adenine (A), guanine (G), cytosine (C), and thymine (T), respectively, for example, compounds 1 to 4. Each of the four nucleotide analogs employs a different fluorescent group. The nucleotide analogs can be recognized by polymerase and incorporated into the growing DNA strand, forming a "template-polymerase-nucleotide analog" complex, and base recognition is achieved through fluorescent signals.

[0048] The sequencing method in this application achieves scarless sequencing through a five-step core process of "binding-recognition-polymerization-cleavage-cycling". The specific steps are as follows:

[0049] Template preparation (S1-S2): The nucleic acid template to be sequenced is immobilized on a chip or microsphere, and the nucleic acid molecular cluster to be tested is formed by bridge amplification;

[0050] Base recognition (S3-S4): Compounds 1 to 4 and polymerase are added to the system. Any one of compounds 1 to 4 is incorporated into the DNA strand and terminates the elongation, forming an intermediate complex. Unreacted compounds are washed away, and the base type is recorded by fluorescence detection. Depending on the fluorescence pattern, a fluorescently labeled active group may be added for coupling reaction or environmental regulation may be required.

[0051] Competitive substitution (S5): Compounds 5 to 8 are added to polymerase. Compounds 5 to 8 competitively replace compounds 1 to 4 in the intermediate complex, forming phosphodiester bonds normally, achieving chain extension, and the blocking group ensures the incorporation of a single base.

[0052] Cutting and cleaning (S6-S7): Add cutting fluid to remove the protecting groups of compounds 5 to 8, generating natural 3'-OH; wash away the replaced compounds 1 to 4 and residual reagents;

[0053] Cyclic sequencing (S8): Repeat steps S3 to S7 to achieve multiple rounds of base sequencing and finally obtain the complete nucleic acid sequence.

[0054] The present application will be further described in detail below through specific embodiments. The following embodiments are only for further illustration of the present application and should not be construed as limiting the present application.

[0055] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0056] Example 1

[0057] This example synthesized a nucleotide analog with the structure shown in Formula 1 and a reversible blocking nucleotide derivative with the structure shown in Formula 2.

[0058] The synthetic route for nucleotide analogs is as follows:

[0059]

[0060] Specific synthesis methods include:

[0061] Synthesis of compound G2 (G1→G2: isobutyrylation reaction)

[0062] 10.0 g (25.5 mmol, 1.0 eq) of G1 (Wuhu Huaren product number: HR-001008007) was placed in a dry 50 mL round-bottom flask, and 100 mL of anhydrous pyridine was added to dissolve it. After cooling to 0 °C in an ice bath, 8.0 eq of TMSCl and 3.6 eq of isobutyric anhydride were added sequentially. The mixture was stirred at 0 °C for 30 min, then heated to room temperature and stirred for another 2.5 h. After the reaction was complete, the reaction solution was quenched in ice water and extracted with dichloromethane (2 × 100 mL). The combined organic phases were washed sequentially with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20:1), and 10.8 g of G2 was finally obtained in 75% yield. Its ESI-MS mass spectrometry showed m / z = 463.1 [M+H]. + (Theoretical precise mass 462.04) Mass spectrometry confirmed that the product structure was consistent with the design.

[0063] Synthesis of compound G3 (G2→G3: TBS hydroxyl protection)

[0064] 4.36 g (9.4 mmol, 1.0 eq) of G2 was dissolved in 60 mL of anhydrous pyridine, and 1.7 g (11.3 mmol, 1.2 eq) of TBSCl was added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, most of the pyridine was removed by vacuum distillation. The residue was quenched in ice water and extracted with dichloromethane (2 × 100 mL). The organic phase was washed with saturated brine, dried, and concentrated. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1:1) to give 5.1 g of G3 in 84% yield. Its ESI-MS mass spectrometry showed m / z = 600.2 [M+Na].+ (Theoretical precise mass 576.13 + 23 = 599.13) Mass spectrometry confirmed that the product structure was consistent with the design.

[0065] 5.1 g (8.86 mmol, 1.0 eq) of G3 was dissolved in 200 mL of anhydrous dichloromethane. After cooling to 0°C in an ice bath, 1.772 g (21.26 mmol, 2.4 eq) of pyridine and 2.53 g (17.72 mmol, 2 eq) of benzoyl chloride were added. The mixture was stirred at 0°C for 30 min, then brought to room temperature and stirred for another 3 h. After the reaction was complete, the mixture was quenched with saturated ammonium chloride solution. The organic phase was extracted with dichloromethane and washed successively with saturated sodium bicarbonate solution and saturated brine. After drying and concentration, the mixture was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 3:1) to obtain 5.4 g of G4 in 78% yield. Its ESI-MS mass spectrometry showed m / z = 681.2 [M+H]. + (Theoretical precise mass 680.15) Mass spectrometry confirmed that the product structure was consistent with the design.

[0066] Synthesis of compound G5 (G4→G5: Sonogashira coupling)

[0067] 5.4 g (7.93 mmol, 1.0 eq) of G4, 2.827 g (15.87 mmol, 2 eq) of alkyne (F3C-CO-NH-CH2-C≡CH), 278 mg (0.05 eq) of Pd(PPh3)2Cl2, and 152 mg (0.1 eq) of CuI were added to a dry Schlenk tube. After constructing an inert atmosphere by purging with argon three times, a mixed solvent of 2 eq of anhydrous triethylamine and 140 mL of DMF was added. The mixture was heated to 35 °C and stirred for 6 h. After cooling, the insoluble catalyst was removed by filtration. The filtrate was concentrated under reduced pressure and dissolved in ethyl acetate. The solution was washed successively with 10% dilute hydrochloric acid and saturated brine. After drying and concentration, the solution was purified by silica gel column chromatography (eluent: dichloromethane / ethyl acetate = 5:1) to obtain 4.08 g of G5 in 58% yield. Its ESI-MS... Mass spectrometry showed m / z = 726.3 [M+Na] + (Theoretical precise mass 703.26 + 23 = 726.26) Mass spectrometry confirmed that the product structure was consistent with the design.

[0068] Synthesis of compound G6 (G5→G6: deprotection from TBS)

[0069] 4.08 g (5.814 mmol, 1.0 eq) of G5 was dissolved in 150 mL of tetrahydrofuran, and 2.856 g (17.442 mmol, 3.0 eq) of Et3N·3HF was added. The mixture was stirred at room temperature for 4 h. After the reaction was complete, saturated sodium bicarbonate solution was added for neutralization. The organic phase was extracted with ethyl acetate and washed with saturated brine. After drying and concentration, the product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15:1). The final product, 3.4 g of the target product G6, was obtained in 85% yield, with ESI-MS mass spectrometry showing m / z = 690.2 [M+H]. + (Theoretical precise mass 689.18) Mass spectrometry confirmed that the product structure was consistent with the design.

[0070]

[0071] Synthesis of compound A2 (A1→A2: TBS hydroxyl protection)

[0072] 8.83 g (23.49 mmol, 1.0 eq) of A1 (Wuhu Huaren product code: HR-001008008) was placed in a dry 50 mL round-bottom flask, and 150 mL of anhydrous pyridine was added to dissolve it. Then, 4.33 g (28.2 mmol, 1.2 eq) of TBSCl was added, and the mixture was stirred at room temperature for 12 h. After the reaction was completed, most of the pyridine was removed by vacuum distillation. The residue was quenched in ice water, and ice water was added to precipitate the solid. The solid was filtered and dried to give 10.36 g of A2 in 90% yield. Its ESI-MS mass spectrometry showed m / z = 513.1 [M+Na]. + (Theoretical precise mass 490.09 + 23 = 513.09) Mass spectrometry confirmed that the product structure was consistent with the design.

[0073] Synthesis of compound A3 (A2→A3: aminobenzoylation)

[0074] 10.36 g (21.16 mmol, 1.0 eq) of A2 was dissolved in 100 mL of anhydrous dichloromethane. After cooling to 0°C in an ice bath, 10 g (126.95 mmol, 6 eq) of pyridine and 8.92 g (63.48 mmol, 3 eq) of benzoyl chloride (BzCl) were added sequentially. The mixture was stirred at 0°C for 30 min, then brought to room temperature and stirred for another 3 h. After the reaction was complete, the reaction solution was quenched in a saturated ammonium chloride solution and extracted with dichloromethane (2 × 200 mL). The combined organic phases were washed sequentially with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1:1) to give 11.53 g of A3 in 78% yield. Its ESI-MS mass spectrometry showed m / z = 699.2 [M+H]. + (Theoretical precise mass 698.14) Mass spectrometry confirmed that the product structure was consistent with the design.

[0075] Synthesis of compound A4 (A3→A4: Sonogashira coupling)

[0076] 11.53 g (16.48 mmol, 1.0 eq) of A3, 6 g (33 mmol, 2 eq) of alkyne (F3C-CO-NH-CH2-C≡CH), 0.97 g (5 mol%) of Pd(PPh3)2Cl2, and 520 mg (10 mol%) of CuI were added to a dry Schlenk tube. After constructing an inert atmosphere by purging with argon three times, 2.0 eq of anhydrous triethylamine and 160 mL of DMF mixed solvent were added, and the mixture was heated to 35 °C and stirred for 6 h. After cooling to room temperature, the insoluble catalyst was removed by filtration. The filtrate was concentrated under reduced pressure and dissolved in ethyl acetate. The organic phase was washed successively with 10% dilute hydrochloric acid and saturated brine. After drying with anhydrous sodium sulfate, the mixture was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / ethyl acetate = 4:1) to give 6.44 g of A4 in 54% yield. Its ESI-MS... Mass spectrometry showed m / z = 744.3 [M+Na] + (Theoretical precise mass 721.25 + 23 = 744.25) Mass spectrometry confirmed that the product structure was consistent with the design.

[0077] Synthesis of compound A5 (A4→A5: deprotection from TBS)

[0078] 6.44 g (8.92 mmol, 1.0 eq) of A4 was dissolved in 60 mL of tetrahydrofuran, and 4.65 g (26.78 mmol, 3.0 eq) of Et3N·3HF was added. The mixture was stirred at room temperature for 6 h. After the reaction was complete, the mixture was neutralized to neutral with saturated sodium bicarbonate solution. The organic phase was extracted with ethyl acetate (2 × 100 mL), washed with saturated brine, dried, and concentrated. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15:1). The target product A5 was finally obtained in 85% yield, with ESI-MS mass spectrometry showing m / z = 608.2 [M+H]. + (Theoretical precise mass 607.17) Mass spectrometry confirmed that the product structure was consistent with the design.

[0079]

[0080] Synthesis of compound C2 (C1→C2: TBS hydroxyl protection)

[0081] 16 g (45.3 mmol, 1.0 eq) of C1 (Wuhu Huaren product code: HR-001009004) was placed in a dry 500 mL round-bottom flask, and 180 mL of anhydrous pyridine was added to dissolve it. Then, 8.20 g (54.4 mmol, 1.2 eq) of TBSCl was added, and the mixture was stirred at room temperature for 12 h. After the reaction was completed, most of the pyridine was removed by vacuum distillation. The residue was quenched in a large amount of ice water, and a solid precipitated. The solid was purified by filtration and pulping, and then dried overnight in a vacuum drying oven to obtain 18.69 g of C2 in 89% yield. Its ESI-MS mass spectrometry showed m / z = 490.1 [M+Na]. + (Theoretical precise mass 467.07 + 23 = 490.07) Mass spectrometry confirmed that the product structure was consistent with the design.

[0082] Synthesis of compound C3 (C2→C3: aminobenzoylation)

[0083] 16.8 g (35.9 mmol, 1.0 eq) of C2 was dissolved in 200 mL of anhydrous dichloromethane. After cooling to 0 °C in an ice bath, 17.2 g (0.216 mol, 6 eq) of pyridine and 15.05 g (107.7 mmol, 3.0 eq) of benzoyl chloride (BzCl) were added sequentially. The mixture was stirred at 0 °C for 30 min, then brought to room temperature and stirred for another 3 h. After the reaction was complete, the reaction solution was quenched in a saturated ammonium chloride solution and extracted with dichloromethane (2 × 250 mL). The combined organic phases were washed sequentially with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 4:1) to give 16.23 g of C3 in 67% yield. Its ESI-MS mass spectrometry showed m / z = 676.2 [M+H]. + (Theoretical precise mass 675.13) Mass spectrometry confirmed that the product structure was consistent with the design.

[0084] Synthesis of compound C4 (C3→C4: Sonogashira coupling)

[0085] 16.23 g (24 mmol, 1.0 eq) of C3, 5.22 g (48 mmol, 2.0 eq) of alkyne (F3C-CO-NH-CH2-C≡CH), 850 mg (0.05 eq) of Pd (PPh3)2Cl2, and 455 mg (0.1 eq) of CuI were added to a dry Schlenk tube. After constructing an inert atmosphere by purging with argon three times, 2.0 eq of anhydrous triethylamine and 180 mL of DMF mixed solvent were added, and the mixture was heated to 40 °C and stirred for 6 h. After cooling to room temperature, the insoluble catalyst was removed by filtration. The filtrate was concentrated under reduced pressure and dissolved in ethyl acetate. The organic phase was washed successively with 10% dilute hydrochloric acid and saturated brine. After drying with anhydrous sodium sulfate, the mixture was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / ethyl acetate = 4:1) to obtain 10.77 g in 64% yield. C4, its ESI-MS mass spectrometry shows m / z=721.3 [M+Na] + (Theoretical precise mass 698.24 + 23 = 721.24) Mass spectrometry confirmed that the product structure was consistent with the design.

[0086] Synthesis of compound C5 (C4→C5: deprotection of TBS)

[0087] 10.77 g (15.4 mmol, 1.0 eq) of C4 was dissolved in 100 mL of tetrahydrofuran, and 8.2 g (46.3 mmol, 3.0 eq) of Et3N·3HF was added. The mixture was stirred at room temperature for 6 h. After the reaction was complete, the mixture was neutralized to neutral with saturated sodium bicarbonate solution. The organic phase was extracted with ethyl acetate (2 × 250 mL), washed with saturated brine, dried, and concentrated. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15:1). 7.37 g of the target product C5 was finally obtained in 82% yield, with ESI-MS mass spectrometry showing m / z = 585.2 [M+H]. + (Theoretical precise mass 584.15) Mass spectrometry confirmed that the product structure was consistent with the design.

[0088]

[0089] Synthesis of compound T2 (T1→T2: TBS hydroxyl protection)

[0090] 20 g (56.5 mmol, 1.0 eq) of T1 (Wuhu Huaren product code: HR-001009003) was placed in a dry 500 mL round-bottom flask, and 200 mL of anhydrous pyridine was added to dissolve it. Then, 10.4 g (67.7 mmol, 1.2 eq) of TBSCl was added, and the mixture was stirred at room temperature for 12 h. After the reaction was completed, most of the pyridine was removed by vacuum distillation. The residue was quenched in a large amount of ice water, and a solid precipitated. The solid was filtered and purified by slurry re-slurrying. Finally, 24.45 g of T2 was obtained in 92% yield, and its ESI-MS mass spectrometry showed m / z = 491.1 [M+Na]. + (Theoretical precise mass 468.06 + 23 = 491.06) Mass spectrometry confirmed that the product structure was consistent with the design.

[0091] Synthesis of compound T3 (T2→T3: hydroxybenzoylation)

[0092] 16.8 g (35.9 mmol, 1.0 eq) of T2 was dissolved in 20 mL of anhydrous dichloromethane. After cooling to 0°C in an ice bath, 5.68 g (71.8 mmol, 2.0 eq) of pyridine and 6.06 g (43.1 mmol, 1.2 eq) of benzoyl chloride (BzCl) were added sequentially. The mixture was stirred at 0°C for 30 min, then brought to room temperature and stirred for another 3 h. After the reaction was complete, the reaction solution was quenched in a saturated ammonium chloride solution and extracted with dichloromethane (2 × 25 mL). The combined organic phases were washed sequentially with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 4:1) to give 18.7 g of T3 in 78% yield. Its ESI-MS mass spectrometry showed m / z = 573.1 [M+H]. + (Theoretical precise mass 572.08) Mass spectrometry confirmed that the product structure was consistent with the design.

[0093] Synthesis of compound T4 (T3→T4: Sonogashira coupling)

[0094] In a dry Schlenk tube, 18.7 g (32.7 mmol, 1.0 eq) of T3, 12.55 g (65.4 mol, 2.0 eq) of alkyne (F3C-CO-NH-CH2-C≡CH), 1.15 g (0.05 eq) of Pd (PPh3)2Cl2, and 623 mg (0.1 eq) of CuI were added. After constructing an inert atmosphere by purging with argon three times, 2.0 eq of anhydrous triethylamine and 200 mL of a 1:1 mixture of DMF were added. The mixture was heated to 40 °C and stirred for 6 h. After cooling to room temperature, the insoluble catalyst was removed by filtration. The filtrate was concentrated under reduced pressure and dissolved in ethyl acetate. The organic phase was washed successively with 10% dilute hydrochloric acid and saturated brine. After drying with anhydrous sodium sulfate, the mixture was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / ethyl acetate = 10:1) at 72%. The yield was 13.5 g T4, and its ESI-MS mass spectrometry showed m / z = 618.2 [M+Na]. + (Theoretical precise mass 595.20 + 23 = 618.20) Mass spectrometry confirmed that the product structure was consistent with the design.

[0095] Synthesis of compound T5 (T4→T5: deprotection from TBS)

[0096] 13.5 g (22.7 mmol, 1.0 eq) of T4 was dissolved in 20 mL of tetrahydrofuran, and 11.86 g (68.1 mmol, 3.0 eq) of Et3N·3HF was added. The mixture was stirred at room temperature for 6 h. After the reaction was complete, the mixture was neutralized to neutral with saturated sodium bicarbonate solution. The organic phase was extracted with ethyl acetate (2 × 250 mL), washed with saturated brine, dried, and concentrated. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15:1). The final product, 9.5 g of T5, was obtained in 81% yield, with ESI-MS mass spectrometry showing m / z = 482.1 [M+H]. + (Theoretical precise mass 481.11) Mass spectrometry confirmed that the product structure was consistent with the design.

[0097]

[0098] Synthesis of compound T6 (T5→T6: pyrophosphorylation reaction)

[0099] 2.04 g (4.24 mmol, 1.0 eq) of T5 was placed in a dry 50 mL round-bottom flask, and 30 mL of trimethyl phosphate was added to dissolve it. After cooling to 0°C in an ice bath, (2.0 eq) of proton sponge (as an acid-binding agent) was added, and the mixture was stirred for 5 min. Then, 1.28 g (5.08 mmol, 1.2 eq) of bis(chlorophosphoryl) ether (Cl2P(O)-OP(O)Cl2) was slowly added. The mixture was stirred at 0°C for 1 h, then heated to room temperature and stirred for another 3 h. After the reaction was complete, the mixture was quenched with ice water. The crude product was purified by reversed-phase chromatography (eluent: dichloromethane / methanol = 25:1), and 1.26 g of T6 was finally obtained in 49% yield. Its ESI-MS mass spectrometry showed m / z = 640.0 [M+H]. + (Theoretical precise mass 639.06) Mass spectrometry confirmed that the product structure was consistent with the design.

[0100] Synthesis of compound T7 (T6→T7: phosphoramidation-oxidation-deprotection)

[0101] 1.26 g (1.97 mmol, 1.0 eq) of T6 was placed in a dry Schlenk tube, and 15 mL of anhydrous acetonitrile was added. Then, 0.6 g (5.92 mmol, 3.0 eq) of triethylamine and 0.28 g (3.98 mmol, 2.0 eq) of 1-H-tetrazole were added sequentially. After stirring until completely dissolved, 0.749 g (2.37 mmol, 1.2 eq) of bis(2-cyanoethyl)-N,N-diisopropylphosphonamide was slowly added, and the reaction was stirred at room temperature for 4 h. The reaction solution was then cooled to 0 °C, and 3.948 mL (2.0 eq) of a 1.0 M tert-butyl hydroperoxide solution in toluene was slowly added dropwise. The mixture was stirred at 0 °C for 1 h, then brought to room temperature and stirred for another 2 h. Finally, 5 mL of concentrated ammonia (NH4OH) was added to the system, and the mixture was stirred at room temperature for 3 h. The 2-cyanoethyl protecting group was removed, followed by concentration under reduced pressure to remove the solvent. After filtration, the product was purified by reversed-phase silica gel column chromatography (eluent: water / acetonitrile = 1:1, containing 0.1% triethylamine). The final yield was 0.366 g of the target product T7 in 34% yield, with ESI-MS mass spectrometry showing m / z = 520.0 [M+H]. + (Theoretical precise mass 519.02) Mass spectrometry confirmed that the product structure was consistent with the design.

[0102] The synthesis of compounds C7 / G8 / A7 was carried out using compounds C5 / G6 / A5 as starting materials and with reference to compound T7.

[0103]

[0104] Synthesis of Compound 1

[0105] In a dry 2 mL reaction tube, 6-ROX-Linker (27.1 mg, 0.0246 mmol) and TSTU (13.1 mg, 0.0513 mmol) (Cyclone product number SL-C018) were added, followed by 0.2 mL of anhydrous DMF. The mixture was stirred at room temperature for 5 min under nitrogen protection until completely dissolved. DIPEA (10.2 μL, 0.0615 mmol) was added, and the mixture was stirred for another 30 min to activate the carboxyl groups. T7 (10.6 mg, 0.0205 mmol) was added to the above reaction solution, and the mixture was stirred at room temperature for 6 h under nitrogen protection (HPLC monitoring of the reaction progress). After the reaction was completed, ultrapure water (1.0 mL) was added to quench the reaction. The reaction mixture was filtered and purified using reverse-phase C-18 preparation (eluent: water / acetonitrile = 1:1, containing 0.1% triethylamine carbonate) to obtain 28.0 mg of the target product.

[0106] Compounds 2 to 4 were synthesized using compounds C7 / G8 / A7 as starting materials. Different fluorescent groups were linked via amide condensation. The fluorescent groups, which were not repeated, were selected from a wide range of sources, including AF488, AF532, AF633, AF680, AF660, AF700, AF647, AF594, AF555, AF568, CY3, CY5, CY5.5, CY7, CY7.5, ROX, R6G, ATTO495, ATTO532, ATTO700, ATTO680, ATTO655, ATTO647N, ATTO594, ATTO Rho101, ATTO590, ATTOThio12, FAM, VIC, TET, JOE, HEX, CAL Fluor Orange560, TAMRA, CAL Fluor Red610, TEXASRED, and CAL Fluor. Red635, iFluor488, iFluor514, iFluor532, iFluor546, iFluor555, iFluor568, iFluor590, iFluor610, iFluor633, iFluor647, iFluor680, iFluor700, iFluor710, Quasar705, Quasar670; high-purity products (HPLC purity ≥95%) were obtained through reverse-phase synthesis. Mass spectrometry confirmed that the product structure was consistent with the design.

[0107] Example 2

[0108] Nucleic acid sequencing was performed using nucleotide analogs and reversible blocking nucleotide derivatives synthesized in Example 1. The sequencing method achieves scarless sequencing through a five-step core process of "binding-recognition-polymerization-cleavage-cycling," the specific steps of which are as follows:

[0109] Sequencing was performed using the Saluseq Nimbo platform.

[0110] Template Preparation (S1-S2): The nucleic acid template to be sequenced (Cialis Ecoli standard library (catalog number SL-Q00100), insert fragment 450-550bp) is immobilized on the chip, and the nucleic acid molecular clusters to be tested are formed by bridge amplification. Amplification and Sequencing Primer Hybridization: The Saluseq Nimbo sequencing kit (catalog number SL-S00333) is used for library amplification and sequencing primer hybridization. This procedure can stably obtain uniform single molecular clusters that meet the requirements of subsequent sequencing.

[0111] Base recognition (S3-S4): Compounds 1 through 4 (each dNTP (A / C / G / T) at a final concentration of 1.5 μM) and polymerase (Number: NEB BST polymerase, final concentration 0.1 μg / μL) were added to the system reaction buffer: 1×Sequencing Buffer (100 mM Tris-HCl, 20 mM CaCl2, pH 8.0). Reaction conditions: Incubation at 60°C for 20 seconds. During the reaction, any one of compounds 1 through 4 was incorporated into the DNA strand and terminated elongation, forming an intermediate complex; unreacted compounds were washed away. Washing method: Washing with 50 mM Tris-HCl buffer containing 0.05% Tween-20, followed by recording the base type by fluorescence detection; washing was repeated after detection.

[0112] Polymerization (S5): Compounds 5 through 8 (specific amounts: CELON - catalog numbers SL-C088, SL-C089, SL-C090, SL-C091), each dNTP, with a final concentration of 2 μM, are added with polymerase (amount: NEB BST polymerase, final concentration 0.1 μg / μL). The reaction system consists of 100 mM Tris-HCl, 20 mM MgCl2, and pH 8.0. Reaction conditions: incubation at 60°C for 40 seconds. During the reaction, compounds 5 through 8 competitively substitute for compounds 1 through 4 in the intermediate complex, forming phosphodiester bonds normally, achieving chain extension, and the blocking groups ensure single-base incorporation.

[0113] Cutting and Cleaning (S6-S7): Add cutting solution (specific formulation: 50 mM Tris-HCl (pH 8.5), 10 mM NaCl, 0.5 mM TCEP (tris(2-carboxyethyl)phosphine); reaction conditions: incubation at 50°C for 120 seconds. Remove the protecting groups of compounds 5 to 8 to generate native 3'-OH; wash away the replaced compounds 1 to 4 and residual reagents. Cleaning method: 1. Wash with 50 mM Tris-HCl buffer containing 0.05% Tween-20.

[0114] Cyclic sequencing (S8): Repeat steps S3 to S7 to perform the next cycle of sequencing, for a total of 150 sequencing cycles.

[0115] Example 3

[0116] Reversible termination nucleotide (NRT) SBS sequencing was performed using the Saluseq Nimbo platform.

[0117] Template Preparation (S1-S2): The nucleic acid template to be sequenced (Cialis Ecoli standard library (catalog number SL-Q00100), insert fragment 450-550bp) is immobilized on the chip, and the nucleic acid molecule cluster to be tested is formed by bridge amplification. Amplification and Sequencing Primer Hybridization: Library amplification and sequencing primer hybridization are performed using the Saluseq Nimbo sequencing kit (catalog number SL-S00333).

[0118] The sequencing reaction cycle was performed by sequentially pumping the pre-wash buffer and polymerization reaction solution from the Saluseq Nimbo Sequencing Kit (catalog number SL-S00333) into the amplified chip to initiate the polymerization reaction. After the polymerization reaction was completed, signal acquisition was performed on the entire chip to determine the type of bases bound to the primer strands on each amplified cluster. After signal acquisition, elution buffer and excision reaction solution were sequentially pumped in for further reaction, followed by washing with elution buffer.

[0119] Repeat the above steps to perform the next sequencing cycle. A total of 150 sequencing cycles are performed.

[0120] The analysis and comparison of Example 2 and Example 3 were performed on the same sample, comparing the phasing value, pre-phasing value, and Q30 of the reaction sequencing quality. The results are shown in Table 1.

[0121] Table 1. Results of tests in Examples 2 and 3

[0122] Q30（%） Phasing value Pre-Phasing value Example 2 93.46 0.10 0.07 Example 3 89.78 0.27 0.13

[0123] Sequencing results showed that using the nucleotide analogs and reversible blocking nucleotide derivatives synthesized in Example 1 for nucleic acid sequencing solved the problem of residual chemical structure "scars" in the linker strand of fluorescent dyes during elution. The phasing and pre-phasing values ​​of Example 2 were lower than those of Example 3. At the same time, the sequencing quality Q30 was also higher than that of Example 3, indicating that its sequencing quality was higher.

[0124] As can be seen from the above embodiments, the nucleotide analogs provided in Embodiment 1 of this application can be used as a substitute for reversible termination nucleotides (NRTs). By changing the sequencing biochemical process, namely through the five-step core process of "binding-recognition-polymerization-cleavage-cycling", scarless sequencing can be achieved (Embodiment 2). This solves the inherent defect of SBS sequencing technology: the problem that the linker strand of fluorescent dye is prone to leaving chemical structural "scars" during the elution process, reducing the phasing and pre-phasing values ​​in the sequencing process, and ultimately reducing the error rate and improving the quality of sequencing data.

[0125] It is understood that the modified phosphodiester bonds in this application include thiophosphates, dithiophosphates, alkyl phosphates, aminophosphates, aniline phosphates, etc.; that is, the O between the first and second phosphate groups at the 5' end of the nucleotides T7 / C7 / G8 / A7 involved in compounds 1-4 can be replaced not only by CH2, but also by NH, S, CF2, CBr2, Se, etc.; the advantage of CH2 substitution is that it is easy to synthesize based on the original O, and the bond formed by PCP cannot be cleaved by DNA polymerase. The modified pentose portion: the oxygen atom can be replaced by a carbon atom or a sulfur atom. The modified pentose portion: the nucleotides in compounds 1-4 of this application contain a 3'-OH group. Similarly, there is a class of dideoxynucleotides (ddNTPs) that do not contain a 3'-OH group, labeled with different fluorescent dyes (nucleotides labeled similar to those in the Sanger method), with the O between the first and second phosphate groups at the 5' end replaced by CH2, which can also be used in this sequencing process. The linker and fluorescent dye can refer to existing technologies.

[0126] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.

Claims

1. A nucleotide analog, characterized in that: The nucleotide analog has the structure shown in Formula 1; Set 1 ; In Formula 1, R1 is adenine A, guanine G, cytosine C, thymine T, uracil U, or analogs of these purines or pyrimidines; R2 / R3 is a fluorescent group, a reactive group, H, or a molecule that increases the ability to bind to the DNA strand; R4 is a monophosphate group or a polyphosphate group, and in the polyphosphate group, the O between the first phosphate group and the second phosphate group at the 5' end is replaced by CH2, NH, S, CF2, CBr2, or Se; L1 is a linker.

2. The nucleotide analogue according to claim 1, characterized in that: The nucleotide analogues include at least one of compound 1, compound 2, compound 3, and compound 4; Compound 1 ; Compound 2 ; Compound 3 ; Compound 4 ; In compounds 1 to 4, L1 is a linker, and Dye1, Dye2, Dye3 and Dye4 are different fluorescent groups.

3. The nucleotide analogue according to claim 2, characterized in that: The fluorescent groups of compounds 1 to 4 are non-repeatingly selected from AF488, AF532, AF633, AF680, AF660, AF700, AF647, AF594, AF555, AF568, CY3, CY5, CY5.5, CY7, CY7.5, ROX, R6G, ATTO495, ATTO532, ATTO700, ATTO680, ATTO655, ATTO647N, ATTO594, ATTO Rho101, ATTO590, ATTO Thio12, FAM, VIC, TET, JOE, HEX, CALFluor Orange560, TAMRA, CAL Fluor Red610, TEXAS RED, and CAL Fluor. Red635, iFluor488, iFluor514, iFluor532, iFluor546, iFluor555, iFluor568, iFluor590, i Fluor610, iFluor633, iFluor647, iFluor680, iFluor700, iFluor710, Quasar705, Quasar670.

4. The nucleotide analogue according to any one of claims 1-3, characterized in that: The connector can be a shardable connector or a non-shardable connector; Optionally, the cleavable linker includes at least one of the following: electrophilic cleavage linker group, nucleophilic cleavage linker group, photolytic linker group, cleavage group under reducing conditions, cleavage group under oxidizing conditions, safety handle type linker group, and group cleaved by elimination mechanism. Optionally, the cleavable linker is at least one selected from alkyl, allyl, azidomethylene, 2-nitrobenzyl, and dithio. Optionally, the non-cleavable linker includes at least one of a polyethylene glycol chain and a polypeptide chain.

5. A reagent kit, characterized in that: Includes the nucleotide analogues according to any one of claims 1-4.

6. The reagent kit according to claim 5, characterized in that: It also includes reversible blocking nucleotide derivatives, which have the structure shown in Formula 2; Formula 2 ; In Formula 2, R5 is a 3'-OH modifying group that can be orthogonally cleaved, i.e., a reversible blocking group; R6 is adenine A, guanine G, cytosine C, thymine T, or uracil U; R7 is a monophosphate group or polyphosphate group that can normally form a phosphodiester bond.

7. The reagent kit according to claim 6, characterized in that: The reversible blocking group includes at least one of methylene azide, allyl, ester, phosphoric acid, hydroxylamine, disulfide bond, photolytic group, 2-nitrobenzyl, and azo compound; Optionally, the reversible blocking group includes at least one of the following groups: 。 8. The reagent kit according to claim 7, characterized in that: The reversible blocking nucleotide derivative includes at least one of compound 5, compound 6, compound 7 and compound 8; Compound 5 ; Compound 6 ; Compound 7 ; Compound 8 ; In compounds 5 to 8, N3 is the methylene azido blocking group.

9. The reagent kit according to any one of claims 6-8, characterized in that: It also includes at least one of polymerase, cutting fluid, and scanning buffer; The cutting fluid contains at least one cutting agent selected from Na2S2O4, THPP, TECP, and DTT, which is used to specifically remove the blocking groups of the reversible blocking nucleotide derivative. The scanning buffer is used to adjust the pH and polarity of the reaction environment to suit the needs of fluorescence excitation or quenching.

10. The use of the nucleotide analogue according to any one of claims 1-4 or the kit according to any one of claims 5-9 in nucleic acid sequencing.

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