WO2019071474A1

WO2019071474A1 - Modified nucleoside or nucleotide

Info

Publication number: WO2019071474A1
Application number: PCT/CN2017/105734
Authority: WO
Inventors: 刘二凯; 陈奥; 章文蔚
Original assignee: 深圳华大智造科技有限公司
Priority date: 2017-10-11
Filing date: 2017-10-11
Publication date: 2019-04-18
Also published as: CN110650968B; CN110650968A

Abstract

The present invention relates to the field of nucleotide sequencing. Specifically, the invention relates to a modified nucleoside or nucleotide, wherein the 3'-OH of said modified nucleoside or said nucleotide is reversibly blocked, and the modified nucleoside or said nucleotid carries a detectable marker. The invention also relates to a kit containing said nucleoside or nucleotide, a method for preparing said nucleoside or nucleotide, and a sequencing method based on said nucleoside or nucleotide.

Description

Modified nucleoside or nucleotide

Technical field

The invention relates to the field of nucleic acid sequencing. In particular, the invention provides a modified nucleoside or nucleotide, the 3'-OH of the modified nucleoside or nucleotide is reversibly blocked, and the modified nucleoside or nucleotide Carry a detectable mark. The invention also relates to a kit comprising the nucleoside or nucleotide, a method of making the nucleoside or nucleotide, and a sequencing method based on the nucleoside or nucleotide.

Background technique

DNA sequencing technology includes the first generation DNA sequencing technology represented by Sanger sequencing, and the second generation DNA sequencing technology represented by Illumina Hiseq 2500, Roche 454, ABI Solid, BGI SEQ-500, and the like. The Sanger sequencing method has the characteristics of simple experimental operation, intuitive and accurate results, and short experimental period. It has wide application in the fields of clinical gene mutation detection and genotyping, which require high timeliness of detection results. However, the disadvantages of the Sanger sequencing method are small throughput and high cost, which limits its application in large-scale gene sequencing.

Compared with the first generation of DNA sequencing technology, the second generation DNA sequencing technology has the characteristics of large sequencing throughput, low cost, high degree of automation and single molecule sequencing. Taking the sequencing technology of Hiseq 2500V2 as an example, an experimental procedure can generate 10-200 G base data, and the average cost per base is less than 1/1000 of the sequencing cost of the Sanger sequencing method, and the obtained sequencing result is obtained. It can be processed and analyzed directly by computer. Therefore, second generation DNA sequencing technology is very suitable for large-scale sequencing.

The most critical part of the second-generation sequencing technology is sequencing by side synthesis. The usual approach is to reversibly block the deoxyribonucleoside triphosphate (dNTP). In short, this process involves: catalysis by polymerase, Incorporating a dNTP with a reversible blocking group and a detectable label into a growing nucleic acid strand, wherein the reversible blocking group protects the 3'-OH of the dNTP, preventing the dNTP that has been incorporated into the nucleic acid strand from passing through 3' -OH reacts with free dNTPs; detectable labels are detected to determine the type of dNTP introduced; thereafter, the blocking groups and detectable labels on the introduced dNTPs are removed and the next round of sequencing is initiated.

At present, there are various groups for reversible blocking of 3'-OH. For example, Illumina blocks the 3'-OH by azide methylene, and after polymerization and detection, the azide is removed using an organic phosphine. Methylene, release 3'-OH, so that the next cycle can be sequenced; Columbia University Jingyue Ju research group uses allyl block 3'-OH, the process uses metal palladium and triphenylphosphine triple sulfonate The ligand of the acid trisodium salt removes the blocking group by a palladium-catalyzed deallyl reaction; in addition, researchers have used the carbonate and sulfur-sulfur bond to reversibly block, in the cut After the sulfur-sulfur bond is broken, the resulting mercapto group can cleave the carbonate to which the 3'-OH is attached, thereby completing the deblocking.

In the method of sequencing while synthesizing, the blocking method is very strict. Among the above blocking methods, only Illumina's blocking method using azide methylene is successfully used for sequencing, and other methods. It faces a variety of problems in use and has not been successfully applied. The reasons for this situation include: 1) DNA sequencing reactions must be carried out in a near neutral aqueous system, which makes many chemical reactions and blocking groups unusable; 2) the groups generated after blocking must be very Stable, and the resection reaction needs to be rapid and efficient, which also eliminates many types of reactions, such as the above carbonate and sulfur-sulfur bond methods, which are difficult to apply due to the fast resection rate; 3) the resection reaction does not affect the sequencing system. For example, the allyl group is used as a blocking group, and it needs to be catalyzed by a palladium metal complex during excision. In practical applications, it may be unsuitable for the chip, and eventually no mature product appears on the market. .

In addition, although the azide methylene blocking technology used by Illumina is relatively mature, there are also some problems, including insufficient stability of the azide, which leads to a decrease in sequencing quality. In order to solve these problems, in the patent application WO2014139596A1, Illumina optimizes the azidomethylene, which increases the stability, but although the resection efficiency can reach 100%, it still requires a higher temperature, and the excision reagent is not good for DNA. influences. In addition, in its patent application US20130085073A1, Illumina uses a phospho-blocked dNTP for sequencing while synthesizing, but it is clearly shown in the patent that only the hydroxyl groups on the phosphoric acid are all protected before being polymerized by the polymerase, and the hydroxyl groups are all The protected phosphate group cannot be cleaved by endonuclease IV. Thus, the method disclosed in the patent comprises: polymerizing a dNTP in which 3'-OH is blocked by a phosphate group, wherein the hydroxyl group on the phosphate group is completely protected, followed by stepwise excision, that is, first removing the phosphate group A hydroxy protecting group on the cleavage, followed by excision of the entire phosphate group by endonuclease IV, wherein the method used to remove a protecting group includes photocleavage, and Ada (methyltransferase) removes the methyl group. The method disclosed in the patent comprises: polymerizing a dNTP in which 3'-OH is blocked by a phosphate group, wherein the phosphate group, although theoretically feasible, is very cumbersome in practical use.

Summary of the invention

In order to solve the above problems, the inventors of the present application have developed a novel 3'-OH reversibly blocked nucleotide which has higher stability, can be efficiently or even completely polymerized by a polymerase, and can be Efficient or even 100% resection without damage to DNA.

Summary of invention

The inventors of the present application have developed a novel modified nucleoside or nucleotide in which 3'-OH is blocked by a blocking group, and the blocking group can be cleaved without destroying the nucleic acid strand. The reagent is excised to form a free 3'-OH. The modified nucleoside or nucleotide also carries a detectable label, and the detectable label is linked to the base in the nucleotide by a linker comprising a phosphodiester bond, and the excision reagent can be utilized (with excision) The excision reagent of the blocking group is the same or different), and the phosphodiester bond is cleaved to remove the detectable label without destroying the nucleic acid strand.

Thus, in one aspect, the application provides a compound having the structure of formula (I),

Wherein R ₁ and R ₃ are each independently selected from

A is selected from the group consisting of phenyl, naphthyl, anthryl and pyridyl;

R _{2 is} selected from the group consisting of a nitro group, a halogenated C _1-4 alkyl group, a halogen, a hydrogen, an aldehyde group,

Wherein Q is independently selected from C _1-4 alkyl;

R _{4 is} selected from -H, a monophosphate group (-PO ₃ H ₂ ), a diphosphate group (-PO ₃ H-PO ₃ H ₂ ), a triphosphate group (-PO ₃ H-PO ₃ H-PO ₃ H ₂ ) and a tetraphosphoric acid group (-PO ₃ H-PO ₃ H-PO ₃ H-PO ₃ H ₂ );

R ₆ is hydrogen or a hydroxyl group;

m and n are each independently selected from 0, 1, 2, 3, 4, 5;

L is a linking group or does not exist;

Base represents a base, such as a purine base or a pyrimidine base, for example, one selected from the group consisting of A, T, U, C, and G;

Label indicates a detectable label, such as a fluorophore;

Blocker indicates a blocking group.

In one aspect, the application also provides a method of making a modified nucleoside or nucleotide as described above.

Based on the modified nucleosides or nucleotides of the present invention, the inventors of the present application have also developed a sequencing method for polynucleotides. In the sequencing method of the present invention, sequencing is performed while synthesizing a polynucleotide in which a target single-stranded polynucleotide is complementary to each other.

Thus, in one aspect, the application provides a method of preparing a growing polynucleotide complementary to a target single-stranded polynucleotide in a sequencing reaction, comprising incorporating a compound as defined above into the complementary of said growth A polynucleotide, wherein the incorporation of the compound prevents any subsequent nucleotides from being introduced into the growing complementary polynucleotide.

In another aspect, the application provides a method of determining the sequence of a single-stranded polynucleotide of interest, comprising: monitoring sequential incorporation of complementary nucleotides, wherein at least one complementary nucleotide that is incorporated is as described above A defined compound, and detecting a detectable label carried by the compound.

In certain embodiments, the method of determining the sequence of a single-stranded polynucleotide of interest comprises:

(a) providing a mixture comprising a duplex, a compound of formula (I), a polymerase, and a excision reagent; said duplex comprising a growing nucleic acid strand and a nucleic acid molecule to be sequenced;

(b) Carry out a reaction cycle comprising the following steps (i), (ii) and (iii):

Step (i): using a polymerase to incorporate the compound into a growing nucleic acid strand to form a nucleic acid intermediate comprising a blocking group and a detectable label;

Step (ii): detecting a detectable label on the nucleic acid intermediate;

Step (iii): The blocking group on the nucleic acid intermediate is removed using a resection reagent.

In certain embodiments, the reaction cycle further comprises the step (iv) of removing the detectable label on the nucleic acid intermediate using a scavenging reagent.

In certain embodiments, the ablation reagents used in steps (iii) and (iv) are the same reagents. In certain embodiments, the ablation reagents used in steps (iii) and (iv) are different reagents.

In one aspect, the invention provides a kit comprising first, second, third and fourth compounds, each of said first, second, third and fourth compounds being of the formula A compound of I) which is a derivative of nucleotides A, (T/U), C and G, respectively, and which has a base complementary pairing ability.

The embodiments of the present invention are explained in detail below in conjunction with the drawings and the detailed description of the invention. However, those skilled in the art will understand that the following drawings and the detailed description of the invention are only intended to illustrate the invention and not to limit the scope of the invention. The various objects and advantageous aspects of the invention will be apparent to those skilled in the <

BRIEF DESCRIPTION OF THE DRAWINGS

1 is a process in which a dTTP having a Cy3-tag and a 3'-OH is blocked is prepared in Example 1, and is involved in HPLC chromatogram of intermediate compound IV-1.

Figure 2 is an HPLC chromatogram of dTTP with Cy3-labeled and 3'-OH blocked in Example 1.

Figure 3 is an HPLC chart of the intermediate compound IV-2 involved in the preparation of dATP with Cy3-labeled and 3'-OH blocked in Example 2.

Fig. 4 exemplarily illustrates the process of determining the polymerization efficiency of dTTP in Example 3.

Fig. 5 exemplarily illustrates the process of determining the excision efficiency of the blocking group in Example 4.

Figure 6 shows the efficiency of the blocking group being cleaved at different excision times in Example 4.

Fig. 7 exemplarily illustrates the process of determining the excision efficiency of the fluorophore in Example 5.

Figure 8 shows the efficiency of fluorophore excision at different cut-off times in Example 5.

Figure 9 is an LC spectrum of dTTP blocked by methyl phosphate in Example 6 after stability testing.

Figure 10 is a LC map of the azide methylene-blocked dTTP in Example 6 after stability testing.

The information of the sequences involved in the present invention is provided in the following table.

序列号(SEQ ID NO:)Serial number (SEQ ID NO:)	描述 description
11	模板template
22	引物Primer
33	引物Primer

Sequence information

Sequence 1 (SEQ ID NO: 1): 33 bp

Sequence 2 (SEQ ID NO: 2): 31 bp

Sequence 3 (SEQ ID NO: 3): 32 bp

Detailed description of the invention

In the present invention, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, unless otherwise defined. In the embodiments of the present invention, methods and materials similar or equivalent to those described herein can be used, and only the exemplary suitable methods and materials are described below. All publications, patent applications, patents, and other references are incorporated herein by reference. Moreover, the materials, methods, and examples are illustrative only and not imaginative. Also, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

As used herein, the term "C _1-4 alkyl" refers to a straight or branched alkyl group having from 1 to 4 carbon atoms, including but not limited to C _1-2 alkyl, C _1-3 Alkyl, C _2-4 alkyl, for example: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl and the like.

As used herein, the term "halo" refers to the replacement of a hydrogen on a group or compound by one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9) halogen atoms, including Fully halogenated and partially halogenated.

As used herein, the term "halogen" includes fluoro, chloro, bromo, iodo.

As used herein, the term "halo C _1-4 alkyl" refers to a radical derived from one or more halogen atoms substituted by one or more hydrogen atoms on a C _1-4 alkyl group, said "halogen" And "C _1-4 alkyl" are as defined above. Halo C _1-4 alkyl includes, but is not limited to, halo C _1-2 alkyl, halo C _1-3 alkyl, halo C _2-4 alkyl, such as halomethyl (eg fluoromethyl) , chloromethyl), haloethyl (eg fluoroethyl, chloroethyl), halopropyl, haloisopropyl, halo-n-butyl, halo sec-butyl, halogenated Isobutyl, halogenated tert-butyl.

As used herein, the term "blocking" refers to the formation of a 3'-OH formation of a nucleoside or nucleotide using a specific group to polymerize a possible polymerase (eg, a DNA polymerase). termination. The group that is used for blocking is referred to as a "blocking group." In certain preferred embodiments, the blocking group can be removed such that the blocked hydroxyl group can be converted to a reactive hydroxyl group, such blockage being referred to as "reversible blocking." The group used for reversible blocking is referred to as a "reversible blocking group."

As used herein, the term "support" refers to any material (solid or semi-solid) that allows for stable attachment of nucleic acids, such as latex beads, dextran beads, polystyrene, polypropylene, polyacrylamide gels, Gold thin layers, glass and silicon wafers. In some exemplary embodiments, the support is optically clear, such as glass. As used herein, "stable attachment" means that the linkage between the nucleic acid molecule and the support is sufficiently strong that the nucleic acid molecule is not subjected to various reaction or treatment conditions (eg, polymerization and washing treatment). Detach the support.

As used herein, the term "connected" is intended to cover any form of linkage, such as covalent linkages and non-covalent linkages. In certain exemplary embodiments, the nucleic acid molecule is preferably linked to the support by a covalent means.

As used herein, the term "fragmentation" refers to the process of converting a large nucleic acid fragment (eg, a large DNA fragment) into a small nucleic acid fragment (eg, a small DNA fragment). In certain embodiments, the term "large nucleic acid fragment" is intended to encompass nucleic acid molecules (eg, DNA) greater than 5 kb, greater than 10 kb, greater than 25 kb, eg, greater than 500 kb, greater than 1 Mb, greater than 5 Mb or greater nucleic acid molecules (eg, DNA) ).

As used herein, the term "end-filled" refers to the process of complementing the ends of a nucleic acid molecule having an overhanging end to form a nucleic acid molecule having a blunt end.

As used herein, the terms "linker" and "linker sequence" are used interchangeably. As used herein, the terms "linker" and "linker sequence" refer to a stretch of oligonucleotide sequences introduced human at the 5' and/or 3' end of a nucleic acid molecule. A connector can typically contain one or more regions for achieving a particular function. Thus, when a linker is introduced artificially at the 5' and/or 3' end of the nucleic acid molecule, the linker will be able to perform the specific function, thereby facilitating subsequent applications. For example, the linker can comprise one or more primer binding regions to facilitate binding of the primers. In some exemplary embodiments, the linker may comprise one or more primer binding regions, such as a primer binding region capable of hybridizing to a primer for amplification, and/or capable of hybridizing to a primer for use in a sequencing reaction. Primer binding region. In certain preferred embodiments, the linker comprises a universal linker sequence capable of hybridizing to a universal primer, for example, a universal linker sequence capable of hybridizing to a universal amplification primer and/or a universal sequencing primer. Thus, the nucleic acid molecule carrying the linker can be conveniently amplified and/or sequenced by using universal amplification primers and/or universal sequencing primers. In some exemplary embodiments, the linker can also comprise a tag or tag sequence.

As used herein, the terms "tag" and "tag sequence" are used interchangeably. As used herein, the terms "tag" and "tag sequence" refer to a segment of an oligonucleotide sequence introduced at the 5' and/or 3' end of a nucleic acid molecule with a particular base sequence. Labels are commonly used to identify/distinguish the source of nucleic acid molecules. For example, different tags can be introduced in different nucleic acid molecules from different sources, whereby when these nucleic acid molecules of different origin are mixed together, each nucleic acid molecule can be accurately determined by the unique tag sequence carried on each nucleic acid molecule. origin of. The tag sequence can have any length, such as 2-50 bp, such as 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 bp, depending on actual needs.

As used herein, the term "hybridization" generally refers to hybridization under stringent conditions. Hybridization techniques are well known in the field of molecular biology. For illustrative purposes, the stringent conditions include, for example, moderate stringency conditions (eg, hybridization at about 45 ° C in 6 x sodium chloride / sodium citrate (SSC), then in 0.2 x SSC / 0.1% SDS One or more washes at about 50-65 ° C; high stringency conditions (eg, hybridization at about 45 ° C in 6 x SSC, then at about 68 ° C in 0.1 x SSC / 0.2% SDS or Multiple washes; and other stringent hybridization conditions known to those skilled in the art (see, for example, Ausubel, FM et al, 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc. New York, pages 6.3.1-6.3.6 and 2.10.3).

As used herein, the expression "reaction system containing a solution phase and a solid phase" means that the reaction system of the present invention comprises both a support and a substance attached to the support (solid phase), and a solution/solvent dissolved in the solution/solvent. The substance in the solution (solution phase). Accordingly, the expression "removing the solution phase of the reaction system" means removing the solution in the reaction system and the substance (solution phase) contained therein, and retaining only the support in the reaction system and the substance attached to the support. Quality (solid phase). In the context of the present invention, the substance (solid phase) attached to the support may comprise a nucleic acid molecule to be sequenced, a growing nucleic acid strand, and/or a duplex formed by the nucleic acid molecule to be sequenced and the growing nucleic acid strand. .

As used herein, the term "primer" refers to an oligonucleotide sequence that hybridizes to a complementary sequence and initiates a specific polymerization reaction. In general, the sequence of the primer is selected/designed to have maximum hybridization activity for the complementary sequence, while having very low non-specific hybridization activity for other sequences, thereby minimizing non-specific amplification. Methods for designing primers are well known to those skilled in the art and can be performed using commercially available software (e.g., Primer Premier version 6.0, Oligo version 7.36, etc.).

As used herein, the term "polymerase" refers to an enzyme capable of performing a nucleotide polymerization reaction. Such an enzyme is capable of introducing a nucleotide paired with a nucleotide at a position corresponding to a template nucleic acid at the 3' end of the growing nucleic acid strand according to the principle of base complementary pairing.

As used herein, the expressions "A, (T/U), C, and G" are intended to cover two instances: "A, T, C, and G" and "A, U, C, and G." Thus, the expression "the four compounds are derivatives of nucleotides A, (T/U), C and G, respectively" is intended to mean that the four compounds are nucleotides A, T, C and G, respectively. Derivatives, or derivatives of nucleotides A, U, C and G, respectively.

As used herein, the expression "a compound having a base complementary pairing ability" means that the compound is capable of pairing with a corresponding base and forming a hydrogen bond according to the principle of base complementary pairing. According to the principle of base complementary pairing, base A can be paired with base T or U, and base G can be paired with base C. Therefore, when a compound having a base complementary pairing ability is a derivative of nucleotide A, it will be able to pair with a base T or U; when a compound having a base complementary pairing ability is a derivative of a nucleotide T or U When it is a substance, it will be able to pair with base A; when a compound having a base complementary pairing ability is a derivative of nucleotide C, it will be able to pair with base G; when a compound having a base complementary pairing ability is In the case of a derivative of nucleotide G, it will be able to pair with base C.

(1) Modified nucleoside or nucleotide and preparation method thereof

Modified nucleoside or nucleotide

Wherein R ₁ and R ₃ are each independently selected from

R _{2 is} selected from the group consisting of nitro, halo C _1-4 alkyl (eg, fluoro C _1-4 alkyl), halogen (eg, fluorine, chlorine, bromine, iodine), hydrogen, aldehyde,

Wherein Q is independently selected from C _1-4 alkyl (eg, C _1-2 alkyl, C _1-3 alkyl, C _2-4 alkyl, such as methyl, ethyl, n-propyl, isopropyl , n-butyl, sec-butyl, isobutyl, tert-butyl);

R ₆ is hydrogen or a hydroxyl group;

m and n are each independently selected from 0, 1, 2, 3, 4, 5;

L is a linking group or does not exist;

Label indicates a detectable label, such as a fluorophore;

Blocker indicates a blocking group.

In the compound of the present invention, A is an aromatic group, and after the phosphodiester bond attached thereto is broken, an aromatic ring having a hydroxyl group can be formed, which is advantageous for the stable existence of the cut product; R ₁ , R ₂ and R ₃ are electron withdrawing The base helps the phosphodiester bond between A and R ₁ to be cleaved by the excision reagent.

In certain embodiments, R _{1 is} selected from

In certain embodiments, R ₁ is

In certain embodiments, R _{2 is} selected from the group consisting of nitro, trifluoromethyl, fluoro, chloro, hydrogen, and aldehyde. In certain embodiments, R ₂ is a nitro group.

In certain embodiments, R _{3 is} selected from

In certain embodiments, R ₃ is

In certain embodiments,

From:

In certain embodiments,

for

The compound of the present invention may be a nucleoside or a nucleotide, and thus R ₄ may be -H, a monophosphate group (-PO ₃ H ₂ ), a diphosphate group (-PO ₃ H-PO ₃ H ₂ ), three Phosphoric acid group (-PO ₃ H-PO ₃ H-PO ₃ H ₂ ) or tetraphosphoric acid group (-PO ₃ H-PO ₃ H-PO ₃ H-PO ₃ H ₂ ).

In certain embodiments, R ₄ is a monophosphate group (-PO ₃ H ₂ ), a diphosphate group (-PO ₃ H-PO ₃ H ₂ ), a triphosphate group (-PO ₃ H-PO ₃ H-PO ₃ H ₂ ) or tetraphosphate group (-PO ₃ H-PO ₃ H-PO ₃ H-PO ₃ H ₂ ) nucleotide. In this case, the compound is a nucleotide.

In certain embodiments, R ₄ is a triphosphate group (-PO ₃ H-PO ₃ H-PO ₃ H ₂ ). In this case, the compound is a nucleoside triphosphate.

In certain embodiments, R ₄ is -H. In this case, the compound is a nucleoside.

Among the compounds of the present invention, Blocker can have a variety of structures. In certain embodiments, the structure of the Blocker is:

Wherein, Ra ₁ and Ra ₂ are each independently selected from the group consisting of H, F, -CF ₃ , -CHF ₂ , -CH ₂ F, -CH ₂ W, -COOW, -CONHW; W is selected from C ₁ -C ₆ alkyl groups. .

In certain embodiments, the structure of the Blocker is:

Wherein Rb ₁ , Rb ₂ , Rb ₃ , Rb ₄ and Rb ₅ are each independently selected from H and C ₁ -C ₆ alkyl.

In certain embodiments, the structure of the Blocker is:

Wherein Rc ₁ and Rc ₂ are each independently selected from the group consisting of H, F, Cl and -CF ₃ .

In certain embodiments, the structure of the Blocker is:

Wherein R _{5 is} selected from C _1-4 alkyl (eg, C _1-2 alkyl, C _1-3 alkyl, C _2-4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-Butyl, sec-butyl, isobutyl, tert-butyl). Thus, in certain embodiments, the compounds of the invention have the structure of formula (I'):

Wherein R _{5 is} selected from C _1-4 alkyl (eg, C _1-2 alkyl, C _1-3 alkyl, C _2-4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-Butyl, sec-butyl, isobutyl, tert-butyl).

In certain embodiments, R ₅ is methyl or ethyl.

In the compounds of the present invention, a detectable label (Label) is linked to a base by a phosphodiester bond; the phosphodiester bond can be cleaved by a scavenging reagent to remove the detectable label from the compound. In certain embodiments, the detectable label is a fluorophore. Examples of fluorophores that can be used in the present invention include, but are not limited to, various known fluorescent labels such as AF532, ALEX-350, FAM, VIC, TET, CAL.

Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, Cy3, Cy3.5, Cy5, Cy5.5, Quasar 705 and so on. Such fluorophores and methods for their detection are well known in the art and can be selected according to actual needs. In certain embodiments, the Label in the compounds of the invention is Cy3, Cy3.5, Cy5 or Cy5.5.

The compounds of the invention may be deoxyribonucleotides, ribonucleotides, deoxyribonucleosides or ribonucleosides. Therefore, R ₆ may be hydrogen or a hydroxyl group. In certain embodiments, R ₆ is hydrogen. In this case, the compound is a deoxyribonucleotide or a deoxyribonucleoside.

Among the compounds of the present invention,

L functions as a linking group in which L may or may not be present. In certain embodiments, m is one. In certain embodiments, n is one. In certain embodiments, L is

In certain embodiments, the compounds of the invention have the structure of formula (II)

In certain embodiments, R ₂ in formula (II) is a nitro group.

In certain embodiments, the Label in Formula (II) is Cy3.

In certain embodiments, the compounds of the invention have the structure:

Method for preparing modified nucleosides or nucleotides

The present application also provides methods of making modified nucleosides or nucleotides as described above. An exemplary preparation process includes steps 1 - 3:

step 1:

Step 1 further includes the following steps:

Step 1-1: Using Compound I as a starting material, an oxidation reaction is carried out to produce a compound II (intermediate).

In certain embodiments, step 1 comprises: adding methanol and tetrazole to acetonitrile, followed by the addition of a solution in which Compound I is dissolved, and stirring at room temperature to replace the diisopropyl group on compound I with a methoxy group on methanol. Amine; then iodine is added and stirred at room temperature to give compound II.

In certain embodiments, the methanol and/or tetrazole is in excess relative to Compound I. In certain embodiments, the molar ratio of methanol to Compound I is from 2 to 5:1, for example, 2:1, 3:1, 4:1, or 5:1, such as 3:1. In certain embodiments, the molar ratio of the tetrazole to Compound I is from 2 to 5:1, for example, 2:1, 3:1, 4:1, or 5:1, such as 3:1.

In certain embodiments, the iodine is added to the reaction system as a solution, for example, a solution comprising water and/or an organic solvent (eg, a solution comprising water, pyridine, and/or tetrahydrofuran).

In certain embodiments, the iodine is in excess relative to Compound I. In certain embodiments, the molar ratio of Compound I to iodine is 1: 1.5-5, such as 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5, for example 1:1.5.

In certain embodiments, step 1-1 further comprises: after the oxidation reaction of Compound I is completed, sodium sulfite is added to remove unreacted iodine.

In certain embodiments, step 1-1 further comprises purifying Compound II.

Step 1-2: Deprotection of Compound II to give Compound III.

In certain embodiments, step 1-2 comprises mixing compound II with trichloroacetic acid and stirring.

In certain embodiments, step 1-2 comprises: adding compound II to a solution comprising trichloroacetic acid (eg, a solution of trichloroacetic acid in dichloromethane), and stirring at room temperature to provide compound III.

In certain embodiments, the trichloroacetic acid is in excess relative to Compound II.

In certain embodiments, steps 1-2 further comprise purifying compound III.

Step 1-3: The compound III is subjected to a triphosphate reaction and a deprotection reaction to produce a compound IV.

In certain embodiments, steps 1-3 comprise: adding 2-chloro-4H-1,3,2-benzophosphono-4- to a solution comprising Compound III under argon-protected conditions. The ketone was stirred at room temperature; then tri-n-butylammonium pyrophosphate and n-butylamine were added and stirred at room temperature; then iodine was added and stirred at room temperature.

In certain embodiments, the solution comprising Compound III further comprises 1,4-dioxane and anhydrous pyridine.

In certain embodiments, the 2-chloro-4H-1,3,2-benzodioxan-4-one is added to the solution in the form of a solution (eg, a 1,4-dioxane solution). In solution of compound III.

In certain embodiments, the molar ratio of compound III to 2-chloro-4H-1,3,2-benzodioxan-4-one is 1:1-2, such as 1:1.0, 1:1.1. 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2.0, for example 1:1.1.

In certain embodiments, the tri-n-butylammonium pyrophosphate and/or n-butylamine is added to the reaction system in the form of a solution (eg, N,N-dimethylformamide solution).

In certain embodiments, the tri-n-butylammonium pyrophosphate is in excess relative to Compound III. In certain embodiments, the molar ratio of Compound III to tri-n-butylammonium pyrophosphate is 1:1.5-5, such as 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1: 4. 1:4.5 or 1:5, for example 1:1.5.

In certain embodiments, the iodine is in excess relative to Compound III. In certain embodiments, the molar ratio of Compound I to iodine is 1: 1.5-5, such as 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5, for example 1:1.5.

In certain embodiments, steps 1-3 further comprise: after the triphosphate reaction of the compound III and the deprotection reaction are completed, sodium sulfite is added to remove unreacted iodine.

In certain embodiments, steps 1-3 further comprise purifying compound IV.

Step 2

Step 2 further includes the following steps:

Step 2-1: 2-Hydroxyacetic acid and N-ethylenediamine trifluoroacetamide are reacted to form Compound V.

In certain embodiments, step 2-1 comprises: adding O-(N-succinimidyl)-N,N,N',N'-tetramethylurea tetrafluoroborate to 2-hydroxyacetic acid and N,N-diisopropylethylamine was stirred at room temperature, then N-ethylenediamine trifluoroacetamide was added and stirred at room temperature to give Compound V.

In certain embodiments, the molar ratio of 2-hydroxyacetic acid to N-ethylenediamine trifluoroacetamide is from 1:1 to 1:2, such as 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2.0, for example 1:1.2.

In certain embodiments, the molar ratio of 2-hydroxyacetic acid to tetrafluoroboric acid O-(N-succinimidyl)-N,N,N',N'-tetramethylurea is 1:1-1. : 2, for example 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2.0, for example 1 :1.2.

In certain embodiments, the molar ratio of 2-hydroxyacetic acid to N,N-diisopropylethylamine is from 1:1 to 1:2, such as 1:1.0, 1:1.1, 1:1.2, 1: 1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2.0, for example 1:1.5.

In certain embodiments, step 2-1 further comprises purifying compound V.

Step 2-2: Compound V is reacted in the presence of N,N-diisopropylethylamine and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite to form compound VI.

In certain embodiments, step 2-2 comprises adding 2-cyanoethyl N,N-diiso to a solution comprising compound V and N,N-diisopropylethylamine at 0 °C. The propyl chlorophosphoramidite was stirred at 0 ° C for a while, slowly warmed to room temperature and stirring was continued to give compound VI.

In certain embodiments, the solution comprising Compound V and N,N-diisopropylethylamine further contains dichloromethane.

In certain embodiments, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite is added as a solution (eg, a solution of dichloromethane).

In certain embodiments, the molar ratio of compound V to N,N-diisopropylethylamine is from 1:1 to 1:2, such as 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2.0, for example 1:1.5.

In certain embodiments, the molar ratio of compound V to 2-cyanoethyl N,N-diisopropylchlorophosphoramidite is from 1:1 to 1:2, such as 1:1.0, 1:1.1, 1 : 1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2.0, for example 1:1.

In certain embodiments, step 2-2 further comprises purifying compound VI.

Step 2-3: Compound VI is subjected to an oxidation reaction to give compound VI'.

In certain embodiments, step 2-3 comprises: mixing compound VI with tetrazole and 2-nitro-5-hydroxy-benzoic acid tert-butyl ester, stirring at room temperature for a period of time, adding iodine, and continuing to stir at room temperature , the compound VI' is produced.

In certain embodiments, the tetrazole is in excess relative to Compound VI. In certain embodiments, the molar ratio of compound VI to tetrazole is 1:5, such as 1:1, 1:2, 1:3, 1:4, or 1:5, such as 1:2.

In certain embodiments, 2-nitro-5-hydroxy-benzoic acid tert-butyl ester is in excess relative to Compound VI. In certain embodiments, the molar ratio of compound VI to 2-nitro-5-hydroxy-benzoic acid tert-butyl ester is 1:5, such as 1:1, 1:2, 1:3, 1:4 or 1 :5, for example 1:2.

In certain embodiments, Compound VI is dissolved in acetonitrile prior to the reaction. In certain embodiments, the tetrazole and 2-nitro-5-hydroxy-benzoic acid tert-butyl ester are combined with Compound VI in the form of a solution (eg, an acetonitrile solution).

In certain embodiments, the iodine is in excess relative to the compound VI. In certain embodiments, the molar ratio of compound VI to iodine is 1: 1.5-5, such as 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5, for example 1:1.5.

In certain embodiments, steps 2-3 further comprise: after the oxidation reaction of Compound VI is complete, sodium sulfite is added to remove unreacted iodine.

Step 2-4: Deprotection of compound VI'.

In certain embodiments, steps 2-4 include: adding aqueous ammonia to compound VI' to remove trifluoroacetyl and cyanoethyl groups; removing aqueous ammonia, and adding potassium hydroxide to remove tert-butyl groups to provide compound VII.

In certain embodiments, steps 2-4 include: adding excess aqueous ammonia to compound VI', stirring at room temperature; removing aqueous ammonia, adding excess potassium hydroxide, and stirring at room temperature.

In certain embodiments, potassium hydroxide is added as a solution (eg, an aqueous solution).

In certain embodiments, steps 2-4 further comprise: purifying compound VII.

Step 3:

Step 3-1: Compound VII is reacted with an N-hydroxysuccinimide ester of Cy3 under weakly basic conditions to give compound VIII.

In certain embodiments, step 3-1 comprises: adding N,N-diisopropylethylamine and compound VII to the N-hydroxysuccinimide ester of Cy3, and stirring at room temperature to provide compound VIII.

In certain embodiments, step 3-1 comprises: adding a DMF solution comprising N,N-diisopropylethylamine and Compound VII to a solution of N-hydroxysuccinimide ester of Cy3 in DMF, room temperature Stirring gives compound VIII.

In certain embodiments, step 3-1 further comprises: purifying compound VIII.

Step 3-2: Reaction of Compound IV with Compound VIII to form a modified nucleoside or nucleotide of the invention.

In certain embodiments, step 3-2 comprises: compound VIII, O-(N-succinimidyl)-N,N,N',N'-tetramethylurea and N,N tetrafluoroborate - Diisopropylethylamine is mixed, and then compound IV is added and stirred at room temperature to obtain a compound of the present invention.

In certain embodiments, the reaction of Step 3-2 is carried out in DMF.

In certain embodiments, Compound IV is added to the reaction system as a solution (eg, a solution comprising sodium bicarbonate).

In certain embodiments, step 3-2 further comprises: purifying the modified nucleoside or nucleotide of the invention.

(two) sequencing method

In certain embodiments, the incorporation of the compound is achieved by a terminal transferase, a terminal polymerase, or a reverse transcriptase.

In certain embodiments, the method comprises: incorporating the compound into a growing complementary polynucleotide using a polymerase.

In certain embodiments, the method comprises: polymerizing a polymerase using a polymerase under conditions that allow the polymerase to undergo nucleotide polymerization, thereby incorporating the compound into the growing complementary polynucleotide 3' end.

In certain embodiments, the blocking group and the detectable label in the compound are removed prior to introduction of the next complementary nucleotide.

In certain embodiments, the blocking group and the detectable label are removed simultaneously.

In certain embodiments, the blocking group and the detectable label are removed sequentially. For example, the blocking group is removed before or after the detectable label is removed.

Step (ii): detecting a detectable label on the nucleic acid intermediate;

In certain embodiments, for example, wherein the at least one complementary nucleotide incorporated is an embodiment of a compound of formula (I'), the method of determining the sequence of a single-stranded polynucleotide of interest comprises:

(1) providing a duplex comprising a growing nucleic acid strand and a nucleic acid molecule to be sequenced, the duplex being linked to a support;

(2) adding a polymerase for performing nucleotide polymerization, and first, second, third, and fourth compounds, thereby forming a reaction system containing a solution phase and a solid phase; wherein the four compounds are respectively Derivatives of nucleotides A, (T/U), C and G, each having the structure represented by the general formula (I') and having a base complementary pairing ability;

(3) performing nucleotide polymerization using a polymerase under conditions allowing the polymerase to undergo nucleotide polymerization, thereby incorporating one of the four compounds into the 3' end of the growing nucleic acid strand;

(4) removing the solution phase of the reaction system of the previous step, leaving the duplex attached to the support, and detecting the signal emitted by the detectable label on the duplex or the growing nucleic acid strand;

(5) adding a scavenging reagent such that the duplex or the growing nucleic acid strand is contacted with a scavenging reagent in a reaction system containing a solution phase and a solid phase; wherein the excision reagent enables the incorporation of the growing nucleic acid strand The phosphodiester bond (1) and/or the phosphodiester bond (2) in the compound at the 3' end are cleaved and do not affect the phosphodiester bond on the duplex backbone;

(6) The solution phase of the reaction system of the previous step is removed.

In certain preferred embodiments, the method further comprises the steps of:

(7) Repeat steps (2)-(6) or steps (2)-(4) one or more times.

Optionally, the washing operation is performed after any of the steps including the removing operation. In certain preferred embodiments, a washing operation is performed between step (4) and step (5). In certain preferred embodiments, after step (6), a washing operation is performed.

In certain embodiments, the duplex is obtained by a method comprising the steps of:

A primer is provided to anneal the primer to the nucleic acid molecule to be sequenced, which serves as the initial growing nucleic acid strand, together with the nucleic acid molecule to be sequenced, forms a duplex attached to the support.

Nucleic acid molecule

In the method of the invention, the nucleic acid molecule to be sequenced may be any nucleic acid molecule of interest. In certain preferred embodiments, the nucleic acid molecule to be sequenced comprises deoxyribonucleotides, ribonucleotides, modified deoxyribonucleotides, modified ribonucleotides, or any combination thereof. In the methods of the invention, the nucleic acid molecule to be sequenced is not limited by its type. In certain preferred embodiments, the nucleic acid molecule to be sequenced is DNA or RNA. In certain preferred embodiments, the nucleic acid molecule to be sequenced can be genomic DNA, mitochondrial DNA, chloroplast DNA, mRNA, cDNA, miRNA, or siRNA. In certain preferred embodiments, the nucleic acid molecule to be sequenced is linear or circular. In certain preferred embodiments, the nucleic acid molecule to be sequenced is double-stranded or single-stranded. For example, the nucleic acid molecule to be sequenced may be single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), or a hybrid of DNA and RNA. In certain preferred embodiments, the nucleic acid molecule to be sequenced is a single stranded DNA. In certain preferred embodiments, the nucleic acid molecule to be sequenced is a double stranded DNA.

In the methods of the invention, the nucleic acid molecule to be sequenced is not limited by its source. In certain preferred embodiments, the nucleic acid molecule to be sequenced can be obtained from any source, for example, any cell, tissue or organism (eg, viruses, bacteria, fungi, plants, and animals). In certain preferred embodiments, the nucleic acid molecule to be sequenced is derived from a mammal (eg, a human, a non-human primate, a rodent or a canine), a plant, a bird, a reptile, a fish, Fungus, bacteria or virus.

Methods for extracting or obtaining nucleic acid molecules from cells, tissues or organisms are well known to those skilled in the art. Suitable methods include, but are not limited to, ethanol precipitation, chloroform extraction, and the like. A detailed description of such methods can be found, for example, in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FMAusubel et al., Guide to Molecular Biology Experiments , 3rd edition, John Wiley & Sons, Inc., 1995. In addition, various commercial kits can be used to extract nucleic acid molecules from a variety of sources, such as cells, tissues or organisms.

In the method of the invention, the nucleic acid molecule to be sequenced is not limited by its length. In certain preferred embodiments, the nucleic acid molecule to be sequenced can be at least 10 bp, at least 20 bp, at least 30 bp, at least 40 bp, at least 50 bp, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1000 bp in length. , or at least 2000bp. In certain preferred embodiments, the nucleic acid molecule to be sequenced may be 10-20 bp, 20-30 bp, 30-40 bp, 40-50 bp, 50-100 bp, 100-200 bp, 200-300 bp, 300-400 bp, 400-500 bp, 500-1000 bp, 1000-2000 bp, or more than 2000 bp. In certain preferred embodiments, the nucleic acid molecule to be sequenced can have a length of 10-1000 bp to facilitate high throughput sequencing.

In certain preferred embodiments, the nucleic acid molecule can be pretreated prior to attaching the nucleic acid molecule to the support. Such pretreatments include, but are not limited to, fragmentation of nucleic acid molecules, complementation of ends, addition of linkers, addition of tags, repair of nicks, amplification of nucleic acid molecules, isolation and purification of nucleic acid molecules, and any combination thereof.

For example, in certain preferred embodiments, nucleic acid molecules can be subjected to fragmentation in order to obtain nucleic acid molecules of suitable length. In the methods of the invention, fragmentation of a nucleic acid molecule (e.g., DNA) can be performed by any method known to those of ordinary skill in the art. For example, fragmentation can be carried out by enzymatic or mechanical means. The mechanical method can be ultrasonic or physical shear. The enzymatic method can be carried out by digestion with a nuclease (for example, deoxyribonuclease) or restriction endonuclease. In certain preferred embodiments, the fragmentation results in an end of the sequence that is not known. In certain preferred embodiments, the fragmentation results in a known end of the sequence.

In certain preferred embodiments, the enzymatic method uses DNase I to fragment a nucleic acid molecule. DNase I is a universal enzyme that non-specifically cleaves double-stranded DNA (dsDNA) to release 5' phosphorylated dinucleotide, trinucleotide and oligonucleotide products. DNase I has optimal activity in buffers containing Mn ²⁺ , Mg ²⁺ and Ca ²⁺ but no other salts, which are commonly used to fragment a large DNA genome into small DNA fragments, followed by The resulting small DNA fragments can be used to construct a DNA library.

The cleavage properties of DNase I will result in random digestion of DNA molecules (ie, no sequence bias) and, when used in the presence of buffers containing manganese ions, produce predominantly blunt-end dsDNA fragments (Melgar, E. And DAGoldthwait.1968. Deoxyribonucleic acid nucleases. II. The effects of metal on the mechanism of action of deoxyribonuclease IJ Biol. Chem. 243: 4409). When genomic DNA is treated with DNase I, three factors can be considered: (i) amount of enzyme used (unit); (ii) digestion temperature (°C); and (iii) incubation time (minutes). Generally, large DNA fragments or whole genomic DNA can be digested with DNase I for 1-2 minutes between 10 ° C and 37 ° C to produce DNA molecules of suitable length.

Thus, in certain preferred embodiments, the nucleic acid molecule of interest (the nucleic acid molecule to be sequenced) is fragmented prior to step (1'). In certain preferred embodiments, the nucleic acid molecules to be sequenced are subjected to fragmentation by enzymatic or mechanical means. In certain preferred embodiments, the nucleic acid molecule to be sequenced by DNase I is fragmented. In certain preferred embodiments, the nucleic acid molecules to be sequenced are subjected to fragmentation by sonication. In certain preferred embodiments, the fragmented nucleic acid molecule is 50-2000 bp in length, such as 50-100 bp, 100-200 bp, 200-300 bp, 300-400 bp, 400-500 bp, 500-1000 bp, 1000-2000 bp. , 50-1500 bp, or 50-1000 bp.

Fragmentation of double-stranded nucleic acid molecules (eg, dsDNA, genomic DNA) can produce nucleic acid fragments having blunt ends or overhangs of one or two nucleotides in length. For example, when genomic DNA (gDNA) is treated by sonication or DNase I, the product may comprise a DNA fragment having a blunt end or overhang. In this case, the end of the nucleic acid molecule having an overhang can be made up using a polymerase to form a nucleic acid molecule having a blunt end to facilitate subsequent applications (e.g., to facilitate ligation of the fragmented nucleic acid molecule to a linker).

Thus, in certain preferred embodiments, after fragmentation of a nucleic acid molecule to be sequenced (e.g., dsDNA), the fragmented nucleic acid molecule is treated with a DNA polymerase to produce a DNA fragment having a blunt end. In certain preferred embodiments, the DNA polymerase can be any known DNA polymerase, such as T4 DNA polymerase, Pfu DNA polymerase, Klenow DNA polymerase. In some cases, the use of Pfu DNA polymerase may be advantageous because Pfu DNA polymerase not only complements the overhangs to form blunt ends, but also has 3'-5' exonuclease activity, Removal of single nucleotide and dinucleotide overhangs to further increase the number of DNA fragments with blunt ends (Costa, GL and MP Weiner. 1994a. Protocols for cloning and analysis of blunt-ended PCR-generated DNA fragments. PCR Methods Appl 3(5): S95; Costa, GLt A. Grafsky and MP Wemer. 1994b. Cloning and analysis of PCR-generated DNA fragments. PCR Methods Appl 3(6): 338; Costa, GL and MPWeiner. 1994c. Polishing with T4or Pfu polymerase increases the efficiency of cloning of PCR products. Nucleic Acids Res. 22(12): 2423).

In certain preferred embodiments, a linker can be introduced at the 5' and/or 3' end of the nucleic acid molecule to be sequenced. In general, the linker is an oligonucleotide sequence and it can be any sequence, of any length. Linkers of suitable length and sequence can be selected by methods well known in the art. For example, a linker ligated to the end of a nucleic acid molecule to be sequenced is typically 5 to 100 nucleotides in length (eg, 5-10 bp, 10-20 bp, 20-30 bp, 30-40 bp, 40-50 bp, 50-100 bp). A relatively short nucleotide sequence between. In certain preferred embodiments, the linker can have a primer binding region. Such primer binding regions can anneal or hybridize to primers and can be used to elicit specific polymerase reactions. In certain preferred embodiments, the linker has one or more primer binding regions. In certain preferred embodiments, the linker has one or more regions that are capable of hybridizing to a primer for amplification. In certain preferred embodiments, the linker has one or more regions that are capable of hybridizing to primers used in the sequencing reaction. In certain preferred embodiments, a linker is introduced at the 5' end of the nucleic acid molecule to be sequenced. In certain preferred embodiments, a linker is introduced at the 3' end of the nucleic acid molecule to be sequenced. In certain preferred embodiments, a linker is introduced at the 5' and 3' ends of the nucleic acid molecule to be sequenced. In some embodiments, the linker comprises A universal linker sequence sufficient to hybridize to a universal primer. In some embodiments, the linker comprises a universal linker sequence that is capable of hybridizing to a universal amplification primer and/or a universal sequencing primer.

In certain preferred embodiments, the tag sequence can be introduced into the nucleic acid molecule to be sequenced, or the tag sequence can be introduced into the linker described above. A tag sequence refers to a segment of an oligonucleotide having a particular base sequence. The tag sequence can have any length, such as 2-50 bp, such as 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 bp, depending on actual needs. In certain preferred embodiments, each nucleic acid molecule to be sequenced is subjected to a tag sequence containing a particular sequence to facilitate discrimination of the source of each nucleic acid molecule to be sequenced. In certain preferred embodiments, the tag sequence can be introduced directly at the 5' and/or 3' end of the nucleic acid molecule to be sequenced. In certain preferred embodiments, a tag sequence can be introduced into the linker and then ligated to the 5' and/or 3' end of the nucleic acid molecule to be sequenced. The tag sequence can be located anywhere in the linker sequence, such as the 5' and/or 3' end of the linker sequence. In certain preferred embodiments, the linker comprises a primer binding region and a tag sequence. In certain further preferred embodiments, the primer binding region comprises a universal linker sequence that is recognized by universal primers, and preferably, the tag sequence can be located at the 3' end of the primer binding region.

In certain preferred embodiments, different tag sequences are used to label/distinguish nucleic acid molecules from different sources. In such embodiments, preferably, the same tag sequence is introduced into a nucleic acid molecule of the same source, and for each nucleic acid source, a unique tag sequence is used. Subsequently, nucleic acid molecules of different origins can be combined to form a library, and the source of each nucleic acid molecule in the library can be identified/differentiated by the unique tag sequence carried on each nucleic acid molecule.

The nucleic acid molecule to be sequenced can be ligated to a linker or tag sequence by methods well known in the art (eg, PCR or ligation reactions). For example, if a part of the sequence of the nucleic acid molecule to be sequenced is known, the nucleic acid molecule to be sequenced by PCR can be carried out using an appropriate PCR primer containing a linker sequence and a sequence capable of specifically recognizing the nucleic acid molecule to be sequenced. Amplification. The amplified product obtained is the nucleic acid molecule to be tested which introduces a linker at the 5' and/or 3' end. In certain embodiments, a nucleic acid molecule can be linked to a linker using a non-specific ligase (eg, T4 DNA ligase). In certain embodiments, a nucleic acid molecule and a linker can be treated with a restriction enzyme such that they have the same cohesive ends, and then the ligase can be used to link nucleic acid molecules and linkers having the same cohesive ends together. Thereby obtaining a nucleic acid molecule linked to the linker.

In certain embodiments, after the nucleic acid molecule and linker are joined together using a ligase, the resulting product may have a nick at the junction. In this case, a polymerase can be used to repair this incision. For example, a DNA polymerase that loses 3'-5' exonuclease activity but exhibits 5'-3' exonuclease activity can have an incision and repair The ability to complex incisions (Hamilton, S. C., J. W. Farchaus and M. C. Davis. 2001. DNA polymerases as engines for biotechnology. BioTechniques 31: 370). DNA polymerases which can be used for this purpose include, for example, polI of Thermoanaerobacter thermosulfuricus, DNA polI of E. coli, and phage phi29. In a preferred embodiment, polI of Bacillus stearothermophilus is used to repair the nick of dsDNA and form unnotched dsDNA.

In certain preferred embodiments, the nucleic acid molecules to be sequenced can also be amplified to increase the amount or copy number of the nucleic acid molecule. Methods for amplifying nucleic acid molecules are well known to those skilled in the art, a typical example of which is PCR. For example, nucleic acid molecules can be amplified using the following methods: (i) polymerase chain reaction (PCR) requiring temperature cycling (see, eg, Saiki et al, 1995. Science 230: 1350-1354), ligase chain Reaction (see, for example, Barany, 1991. Proc. Natl. Acad. Sci. USA 88: 189-193; Barringer et al, 1990. Gene 89: 117-122), and transcription-based amplification (see, for example, Kwoh et al. Human, 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177); (ii) Isothermal amplification system (see, for example, Guatelli et al., 1990. Proc. Natl. Acad. Sci. USA 87:1874- 1878); QP replicase system (see, eg, Lizardi et al, 1988. BioTechnology 6: 1197-1202); and strand displacement amplification (Nucleic Acids Res. 1992 Apr 11; 20(7): 1691-6). In certain preferred embodiments, the nucleic acid molecule to be sequenced is amplified by PCR, and the primers used for PCR amplification comprise a linker sequence and/or a tag sequence. The PCR product thus produced will carry a linker sequence and/or a tag sequence, which can be conveniently used for subsequent applications (e.g., high throughput sequencing).

In certain preferred embodiments, the nucleic acid molecules to be sequenced are also isolated and purified before or after various pretreatment steps. Such separation and purification steps may be advantageous. For example, in certain preferred embodiments, the isolation and purification steps can be used to obtain nucleic acid molecules of a suitable length (eg, 50-1000 bp) to be sequenced for subsequent applications (eg, high throughput sequencing). In certain preferred embodiments, agarose gel electrophoresis can be utilized to separate and purify the nucleic acid molecules to be sequenced. In certain preferred embodiments, the nucleic acid molecules to be sequenced can be isolated and purified by size exclusion chromatography or sucrose settling.

It should be understood that the pre-treatment steps described above (eg, fragmentation, end-filling, addition of linkers, tag addition, nick repair, amplification, separation, and purification) are merely exemplary and not limiting. Those skilled in the art can perform various desired pretreatments on the nucleic acid molecules to be sequenced according to actual needs, and each pretreatment step is not limited by a specific order. For example, in certain embodiments, the nucleic acid molecule can be first fragmented and a linker added prior to amplification. In other embodiments, the nucleic acid molecule can be amplified first, Then fragment and add the linker. In certain embodiments, the nucleic acid molecule is fragmented and a linker is added without an amplification step.

In certain exemplary embodiments, prior to step (1'), the nucleic acid molecule of interest (e.g., genomic DNA) is subjected to the following pretreatment:

(i) fragmenting the nucleic acid molecule of interest (eg, a large nucleic acid fragment, eg, genomic DNA) to produce a fragmented nucleic acid molecule;

(ii) linking the fragmented nucleic acid molecule to a linker sequence comprising, for example, a primer binding region capable of hybridizing to a universal amplification primer, a primer binding region capable of hybridizing to a universal sequencing primer, and/or a tag sequence, and Optionally performing isolation, purification, and denaturation to produce a nucleic acid molecule to be sequenced;

(iii) linking the nucleic acid molecule to be sequenced to a support to obtain a nucleic acid molecule to be sequenced attached to the support.

Support

In the usual case, the support for ligation of the nucleic acid molecule to be sequenced is in a solid phase for ease of handling. Thus, in the present disclosure, "support" is sometimes also referred to as "solid support" or "solid support." However, it should be understood that the "support" referred to herein is not limited to a solid, it may also be a semi-solid (eg, a gel).

In the method of the present invention, the support for ligation of the nucleic acid molecule to be sequenced may be made of various suitable materials. Such materials include, for example, inorganics, natural polymers, synthetic polymers, and any combination thereof. Specific examples include, but are not limited to, cellulose, cellulose derivatives (such as nitrocellulose), acrylic resins, glass, silica gel, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide, and Crosslinked polyphenylene bromide such as vinyl benzene (see, for example, Merrifield Biochemistry 1964, 3, 1385-1390), polyacrylamide, latex, dextran, rubber, silicon, plastic, natural sponge, metal plastic, cross-linking dextran (e.g., Sephadex ^TM), agarose gel (Sepharose ^TM), and other supports known to the skilled person.

In certain preferred embodiments, the support for ligation of the nucleic acid molecule to be sequenced may be a solid support comprising an inert substrate or matrix (eg, slides, polymer beads, etc.), said inert substrate or matrix Functionalization has been functionalized, for example, by the use of intermediate materials containing reactive groups that allow covalent attachment of biomolecules such as polynucleotides. Examples of such supports include, but are not limited to, polyacrylamide hydrogels supported on an inert substrate such as glass, in particular polyacrylamide hydrogels as described in WO 2005/065814 and US 2008/0280773, wherein The content of the patent application is hereby incorporated by reference in its entirety. In such an implementation A biomolecule (eg, a polynucleotide) can be directly covalently attached to an intermediate material (eg, a hydrogel), while the intermediate material itself can be non-covalently attached to a substrate or matrix (eg, a glass substrate) . In certain preferred embodiments, the support is a slide or wafer having a surface modified with a layer of avidin, amino, acrylamide silane or aldehyde based chemical groups.

In the present invention, the support or solid support is not limited by its size, shape and configuration. In some embodiments, the support or solid support is a planar structure, such as a slide, chip, microchip, and/or array. The surface of such a support may be in the form of a planar layer.

In some embodiments, the support or surface thereof is non-planar, such as the inner or outer surface of a tube or container. In some embodiments, the support or solid support comprises microspheres or beads. As used herein, "microsphere" or "bead" or "particle" or grammatical equivalent refers to a small discrete particle. Suitable bead ingredients include, but are not limited to, plastics, ceramics, glass, polystyrene, methyl styrene, acrylic polymers, paramagnetic materials, cerium oxide sol, carbon graphite, titanium dioxide, latex, cross-linked dextran such as Sepharose , cellulose, nylon, crosslinked micelles and teflon, as well as any other materials outlined herein for the preparation of solid supports. In addition, the beads may be spherical or non-spherical. In some embodiments, spherical beads can be used. In some embodiments, irregular particles can be used. In addition, the beads can also be porous.

In certain preferred embodiments, the support for ligation of the nucleic acid molecule to be sequenced is an array of beads or wells (also referred to as a chip). The array can be prepared using any of the materials outlined herein for preparing a solid support, and preferably, the surface of the beads or pores on the array is functionalized to facilitate ligation of the nucleic acid molecules. The number of beads or holes on the array is not limited. For example, each array may comprise 10-10 ² , 10 ² -10 ³ , 10 ³ -10 ⁴ , 10 ⁴ -10 ⁵ , 10 ⁵ -10 ⁶ , 10 ⁶ -10 ⁷ , 10 ⁷ -10 ⁸ , 10 ⁸ ^-109 or more beads or holes. In certain exemplary embodiments, the surface of each bead or well can be joined to one or more nucleic acid molecules. Correspondingly, each array can be connected 10-10 ² , 10 ² -10 ³ , 10 ³ -10 ⁴ , 10 ⁴ -10 ⁵ , 10 ⁵ -10 ⁶ , 10 ⁶ -10 ⁷ , 10 ⁷ -10 ⁸ , 10 ⁸ - 10 ⁹ or more nucleic acid molecules. Thus, such arrays can be used particularly advantageously for high throughput sequencing of nucleic acid molecules.

As is generally known in the art, a variety of techniques can be utilized to make the support. Such techniques include, but are not limited to, photolithography, stamping techniques, plastic film technology, and microetching techniques. As will be appreciated by those skilled in the art, the techniques used will depend on the composition, structure and shape of the support.

The connection of the nucleic acid molecule to be sequenced to the support

In the methods of the invention, the nucleic acid molecule to be sequenced can be linked (e.g., covalently or non-covalently linked) to the support by any method known to those of ordinary skill in the art. For example, by covalent connection, or by The irreversible passive adsorption, or the intermolecular affinity (eg, the affinity between biotin and avidin), connects the nucleic acid molecule to be sequenced to the support. Preferably, however, the linkage between the nucleic acid molecule to be sequenced and the support is sufficiently strong that the nucleic acid molecule does not detach from the support due to the conditions used in the various reactions (e.g., polymerization) and the washing of the water or buffer solution.

For example, in certain preferred embodiments, the 5' end of the nucleic acid molecule to be sequenced carries a device capable of covalently attaching the nucleic acid molecule to a support, such as a chemically modified functional group. Examples of such functional groups include, but are not limited to, a phosphate group, a carboxylic acid molecule, an aldehyde molecule, a thiol, a hydroxyl group, a dimethoxytrityl group (DMT), or an amino group.

For example, in certain preferred embodiments, the 5' end of the nucleic acid molecule to be sequenced may be modified with a chemical functional group (eg, a phosphoric acid, a thiol or an amino group), and the support (eg, a porous glass bead) may be an amino-alkane. Oxysilane (for example, aminopropyltrimethoxysilane, aminopropyltriethoxysilane, 4-aminobutyltriethoxysilane, etc.) is derivatized so that the nucleic acid can be exchanged by a chemical reaction between the reactive groups The molecule is covalently attached to the support. In certain preferred embodiments, the 5' end of the nucleic acid molecule to be sequenced can be modified with a carboxylic acid or an aldehyde group, and the support (eg, latex beads) is derivatized with hydrazine so that the chemistry between the reactive groups can be The reaction covalently attaches the nucleic acid molecule to the support (Kremsky et al., 1987).

Alternatively, a cross-linking agent can be used to link the nucleic acid molecule of interest to the support. Such crosslinking agents include, for example, succinic anhydride, phenyl diisothiocyanate (Guo et al., 1994), maleic anhydride (Yang et al., 1998), 1-ethyl-3-(3-di). Methylaminopropyl)-carbodiimide hydrochloride (EDC), m-maleimidobenzoic acid-N-hydroxysuccinimide ester (MBS), N-succinimidyl group [4] -iodoacetyl]aminobenzoic acid (SIAB), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide (SMCC), N-γ-maleyl Iminobutyryloxy-succinimide ester (GMBS), 4-(p-maleimidophenyl)butyric acid succinimide (SMPB), and corresponding thio compounds (water soluble) of).

In addition, the support can also be derivatized with a bifunctional crosslinker such as a homobifunctional crosslinker and a heterobifunctional crosslinker to provide a modified functionalized surface. Subsequently, a nucleic acid molecule having a 5'-phosphate, thiol or amino group is capable of interacting with a functionalized surface to form a covalent linkage between the nucleic acid and the support. A large number of bifunctional crosslinkers and methods of use thereof are well known in the art (see, for example, Pierce Catalog and Handbook, pages 155-200).

Primer

In the method of the present invention, the primer may be of any length and may comprise any sequence or any base as long as it can specifically anneal to a region of the target nucleic acid molecule. In other words, in the method of the present invention, Primers are not limited by their length, structure and composition. For example, in some exemplary embodiments, the primers may be 5-50 bp in length, such as 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50 bp. In some exemplary embodiments, the primers are capable of forming a secondary structure (eg, a hairpin structure). In some exemplary embodiments, the primer does not form any secondary structure (eg, a hairpin structure). In some exemplary embodiments, the primers may comprise naturally occurring or non-naturally occurring nucleotides. In some exemplary embodiments, the primer comprises or consists of a naturally occurring nucleotide. In some exemplary embodiments, the primer comprises a modified nucleotide, such as a locked nucleic acid (LNA). In some exemplary embodiments, the primer is capable of hybridizing to a nucleic acid of interest under stringent conditions, such as moderately stringent conditions or highly stringent conditions. In some exemplary embodiments, the primer has a sequence that is fully complementary to a target sequence in a nucleic acid molecule of interest. In some exemplary embodiments, the primer is partially complementary to a target sequence in a nucleic acid molecule of interest (eg, a mismatch is present). In some exemplary embodiments, the primer comprises a universal primer sequence. In some exemplary embodiments, the nucleic acid molecule to be sequenced comprises a linker, and the linker comprises a sequence capable of hybridizing to a universal primer, and the primer used is a universal primer.

Polymerase

In the method of preparing a polynucleotide of the present invention or the sequencing method, a suitable polymerase can be used for nucleotide polymerization. In some exemplary embodiments, the polymerase is capable of synthesizing a new DNA strand (eg, a DNA polymerase) using DNA as a template. In some exemplary embodiments, the polymerase is capable of synthesizing a new DNA strand (eg, a reverse transcriptase) using RNA as a template. In some exemplary embodiments, the polymerase is capable of synthesizing a new RNA strand (eg, RNA polymerase) using DNA or RNA as a template. Accordingly, in certain preferred embodiments, the polymerase is selected from the group consisting of a DNA polymerase, an RNA polymerase, and a reverse transcriptase. A suitable polymerase can be selected for nucleotide polymerization according to actual needs. In certain preferred embodiments, the polymerization reaction is a polymerase chain reaction (PCR). In certain preferred embodiments, the polymerization reaction is a reverse transcription reaction.

In the method of the present invention, nucleotide polymerization can be carried out using KOD polymerase or a mutant thereof. KOD polymerase or a mutant thereof (e.g., KOD POL151, KOD POL157, KOD POL171, KOD POL174, KOD POL376, KOD POL391) has acceptable polymerization efficiency for the modified nucleoside or nucleotide of the present invention. KOD POL391 and KOD POL171 have acceptable polymerization efficiencies for the modified nucleotides of the invention. In certain embodiments, KOD POL391 or KOD POL171 has a polymerization efficiency for the modified nucleotides of the invention of greater than 70%, such as from 70% to 80%, from 80% to 90%, or from 90% to 100%.

Further, as described above, in the method of the present invention, steps (2) - (6) or steps (2) - (4) may be repeated. Thus, in certain preferred embodiments of the invention, one or more rounds of nucleotide polymerization can be carried out. In other words, in certain preferred embodiments of the invention, the nucleotide polymerization can be carried out in one or more steps. In this case, the same or different polymerases can be used for each round of nucleotide polymerization. For example, a first DNA polymerase can be used in the first round of nucleotide polymerization, and a second DNA polymerase can be used in the second round of nucleotide polymerization. However, in certain exemplary embodiments, the same polymerase (eg, the same DNA polymerase) is used in all nucleotide polymerizations.

Polymerization condition

In the method of preparing a polynucleotide of the present invention or the sequencing method, the polymerization of nucleotides is carried out under suitable conditions. Suitable polymerization conditions include the composition of the solution phase and the concentration of each component, the pH of the solution phase, the polymerization temperature, and the like. Polymerization under suitable conditions is advantageous in obtaining acceptable, even high, polymerization efficiencies.

In certain embodiments, the solution phase in which the polymerization occurs comprises monovalent salt ions (eg, sodium ions, chloride ions) and/or divalent salt ions (eg, magnesium ions, sulfate ions). In certain embodiments, the concentration of the monovalent salt or divalent salt ion in the solution phase is 1-200 mM, such as 1 mM, 3 mM, 10 mM, 20 mM, 50 mM, 100 mM, 150 mM, or 200 mM.

In certain embodiments, the solution phase in which the polymerization occurs comprises a buffer solution, such as a buffer solution comprising Tris. In certain embodiments, the concentration of Tris in the solution phase is from 10 mM to 200 mM, such as 10 mM, 20 mM, 50 mM, 100 mM, 150 mM, or 200 mM.

In certain embodiments, the solution phase in which the polymerization occurs comprises an organic solvent such as DMSO or glycerol (glycerol). In certain embodiments, the organic solvent has a mass content in the solution phase of from 0.01% to 10%, such as 0.01%, 0.02%, 0.05%, 1%, 2%, 5%, or 10%.

In certain embodiments, the pH of the solution phase in which the polymerization occurs is from 7.0 to 9.0, such as 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4. , 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.

In certain embodiments, the solution phase in which the polymerization occurs comprises: a monovalent salt ion (eg, sodium ion, chloride ion), a divalent salt ion (eg, magnesium ion, sulfate ion), a buffer solution (eg, a buffer containing Tris) Solution) and organic solvents (such as DMSO or glycerol). In certain embodiments, the pH of the solution phase is 7.8.

In certain embodiments, the polymerization is at 50-65 ° C (eg, 50 ° C, 51 ° C, 52 ° C, 53 ° C, 54 ° C, 55 ° C, 56 ° C, 57 ° C, 58 ° C, 59 ° C, 60 ° C, 61 It is carried out at ° C, 62 ° C, 63 ° C, 64 ° C or 65 ° C).

The time during which the polymerization is carried out can be determined according to actual needs, for example, 1 min to 5 min, 5 min to 10 min, 10 min to 30 min or 30 min to 1 h.

Base derivative

In the method of the present invention, the four compounds used in the step (2) are derivatives of nucleotides A, (T/U), C and G, respectively. In certain exemplary embodiments, the four compounds are derivatives of ribose or deoxyribonucleotides A, T, C, and G, respectively. In certain exemplary embodiments, the four compounds are derivatives of ribose or deoxyribonucleotides A, U, C, and G, respectively. It is particularly advantageous that the four compounds do not undergo a chemical reaction with each other during the nucleotide polymerization.

In addition, the four compounds described have a base complementary pairing ability. For example, when the compound is a derivative of nucleotide A, it will be capable of pairing with the base T or U. When the compound is a derivative of nucleotide T or U, it will be able to pair with base A. When the compound is a derivative of nucleotide C, it will be able to pair with base G. When the compound is a derivative of nucleotide G, it will be able to pair with base C. Thus, in step (3), the polymerase (eg, DNA polymerase) will incorporate a compound capable of complementary pairing with a base at a corresponding position in the template nucleic acid into the growing nucleic acid strand according to the principle of base complementary pairing. 'end. Correspondingly, after determining the type of compound incorporated into the 3' end of the growing nucleic acid strand by a signal emitted by a detectable label (eg, a fluorophore), the base at the corresponding position in the template nucleic acid can be determined by the principle of base complementary pairing. type. For example, if a compound incorporated at the 3' end of the growing nucleic acid strand is identified as a derivative of nucleotide A, then the base at the corresponding position in the template nucleic acid can be determined to be T or U. If a compound incorporated at the 3' end of the growing nucleic acid strand is identified as a derivative of nucleotide T or U, then it is determined that the base at the corresponding position in the template nucleic acid is A. If a compound incorporated at the 3' end of the growing nucleic acid strand is identified as a derivative of nucleotide C, then it is determined that the base at the corresponding position in the template nucleic acid is G. If a compound incorporated at the 3' end of the growing nucleic acid strand is identified as a derivative of nucleotide G, then it is determined that the base at the corresponding position in the template nucleic acid is C.

In the present invention, the hydroxyl group (-OH) at the 3' position of the ribose or deoxyribose of the four compounds is protected. In other words, the hydroxyl groups (-OH) at the 3' position of the ribose or deoxyribose of the four compounds are protected by a protecting group, whereby they are capable of terminating the polymerization of a polymerase such as a DNA polymerase. For example, when any of the four compounds is introduced into the 3' end of the growing nucleic acid strand, due to the ribose or There is no free hydroxyl group (-OH) at the 3' position of the deoxyribose, and the polymerase will not be able to proceed to the next round of polymerization, so that the polymerization will be terminated. In this case, in each round of polymerization, one and only one base will be incorporated into the growing nucleic acid strand.

Furthermore, in certain embodiments, the protecting group at the 3' position of the ribose or deoxyribose of the four compounds can be removed. In step (5), the protecting group is removed and converted to a free hydroxyl group (-OH). Subsequently, the polymerase and the four compounds can be used to carry out the next round of polymerization of the grown nucleic acid strand and introduce one more base.

Thus, in certain embodiments, the four compounds used in step (2) are reversibly blocked: when they are incorporated into the 3' end of the growing nucleic acid strand (eg, in step (3)), they The polymerase will be terminated to continue the polymerization, terminating the further extension of the growing nucleic acid strand; and, after the blocking group they contain is removed, the polymerase will continue to polymerize the growing nucleic acid strand and continue to extend the nucleic acid chain.

Determination of a compound incorporated into a growing nucleic acid strand

In the sequencing method of the present invention, a detectable signal in a duplex or a growing nucleic acid strand is detected after each round of polymerization. By detection, the type of compound incorporated into the growing nucleic acid strand can be determined to determine the type of base at the corresponding position in the nucleic acid molecule to be sequenced.

In certain preferred embodiments, the detectable label is a fluorophore.

In certain preferred embodiments, the sequencing method of the present invention further comprises, after step (4), based on the principle of base complementary pairing, according to the type of compound incorporated in the 3' end of the growing nucleic acid strand in step (3) Determining the type of base at the corresponding position in the nucleic acid molecule to be sequenced. For example, if a compound incorporated at the 3' end of the growing nucleic acid strand is identified as a derivative of nucleotide A, then it is determined that the base at the corresponding position in the nucleic acid molecule to be sequenced is a derivative capable of binding to nucleotide A Paired bases (eg T or U).

More specifically, if a compound incorporated at the 3' end of the growing nucleic acid strand is identified as a derivative of nucleotide A, then it is determined that the base at the corresponding position in the nucleic acid molecule to be sequenced is T or U. If the compound incorporated at the 3' end of the growing nucleic acid strand is determined to be a derivative of nucleotide T or U, then it is determined that the base at the corresponding position in the nucleic acid molecule to be sequenced is A. If the compound incorporated at the 3' end of the growing nucleic acid strand is determined to be a derivative of nucleotide C, then it is determined that the base at the corresponding position in the nucleic acid molecule to be sequenced is G. If the compound incorporated at the 3' end of the growing nucleic acid strand is determined to be a derivative of nucleotide G, then it is determined that the base at the corresponding position in the nucleic acid molecule to be sequenced is C.

Processing of duplexes or growing nucleic acid strands

In the sequencing method of the present invention, each round of polymerization may involve one signal detection, and, in addition to the last round of polymerization, each round of polymerization may involve excision of the duplex or growing nucleic acid strand. deal with. After the last round of polymerization, the duplex or the growing nucleic acid strand may be excised or not excised.

In certain embodiments, the treatment in step (5) can be used to remove blocking groups in the compound incorporated into the 3' end of the growing nucleic acid strand (so that a new round of polymerization can begin) and remove the double A detectable label that may be carried on the chain or growing nucleic acid strand (so as to avoid interference with subsequent detection).

In certain embodiments, the excision reagent for excising the blocking group and the excision reagent for excising the detectable label are the same reagent, or, although different reagents, can be excised under the same conditions, It does not cause mutual interference between the two resection reactions. Therefore, in the step (5), the excision of the blocking group and the excision of the detectable label can be simultaneously performed.

In certain embodiments, the excision reagent for excising the blocking group and the excision reagent for excising the detectable label are different reagents, and the excision needs to be performed under different conditions. In order to avoid mutual interference between the excision reactions, excision of the blocking group and excision of the detectable label can be performed stepwise.

In certain embodiments, for example, wherein the at least one complementary nucleotide incorporated is an embodiment of a compound of formula (I'), step (5) comprises:

Step (5-1): adding a first excision reagent, contacting the duplex or the grown nucleic acid strand with the first excision reagent in a reaction system containing a solution phase and a solid phase without affecting the duplex a phosphodiester bond (1) in a compound incorporated at the 3' end of the growing nucleic acid strand under conditions of a phosphodiester bond on the backbone;

Step (5-2): adding a second excision reagent, so that the duplex or the grown nucleic acid strand is contacted with the first excision reagent in a reaction system containing a solution phase and a solid phase without affecting the duplex The phosphodiester bond (2) in the compound incorporated at the 3' end of the growing nucleic acid strand is cleaved under the condition of a phosphodiester bond on the backbone.

It should be noted that there is no fixed sequence between step (5-1) and step (5-2), and step (5-1) may be performed first, or step (5-2) may be performed first. The use of "first" and "second" merely distinguishes between excising agents and does not represent the order of use of the two excising agents.

The time during which the excision reaction is performed can be determined according to actual needs, for example, 1 min to 5 min, 5 min to 10 min, 10 min to 30 min, or 30 min to 1 h.

Resection reagent

In the present invention, a blocking group and/or a detectable label in the compound of the formula (I) is removed using a scavenging agent. When the compound of the formula (I') is used, the excision reagent of the present invention is capable of cleaving the phosphodiester bond (1) and/or the phosphodiester bond (2) in the compound of the formula (I'), and Does not break the phosphodiester bond in the nucleic acid strand backbone to maintain the integrity of the nucleic acid strand backbone.

In the present invention, excision reagents that can be used to remove blocking groups include, but are not limited to, endonuclease IV. The endonuclease IV can selectively cleave the phosphodiester bond (1) in the compound represented by the formula (I') without affecting the phosphodiester bond in the nucleic acid strand skeleton.

In the present invention, excision reagents that can be used to remove detectable labels include, but are not limited to, alkaline phosphatase. The alkaline phosphatase can selectively cleave the phosphodiester bond (2) in the compound represented by the formula (I') without affecting the phosphodiester bond in the nucleic acid strand skeleton.

The conditions of the excision reaction may depend on the excision reagent. For example, the excision reagent used is a commercially available enzyme, and thus, the conditions of the excision reaction can be determined according to the use conditions recommended by the supplier (for example, a recommended buffer solution, temperature, etc.). .

Washing step

In the sequencing method of the present invention, the washing step can be increased as needed. The washing step can be increased at any desired stage, and optionally, the washing step can be performed one or more times.

For example, in the step (4), after removing the solution phase of the reaction system, one or more washings may be performed to sufficiently remove the residual solution phase. Such a washing step may be advantageous in that it can be used to substantially remove free (ie, unincorporated growth nucleic acid strands) compounds that carry detectable labels, minimizing non-specific signals.

Similarly, in the step (6), after removing the solution phase of the reaction system, one or more washings may be performed to sufficiently remove the residual solution phase. Such a washing step may be advantageous, which can be used to sufficiently remove the ablation reagent applied in step (5), thereby minimizing the adverse effects on subsequent reactions.

The washing step can be carried out using a variety of suitable washing solutions. Examples of such wash solutions include, but are not limited to, phosphate buffer, citrate buffer, Tris-HCl buffer, acetate buffer, carbonate buffer, and the like. It is within the ability of those skilled in the art to select a suitable wash solution (including suitable ingredients, concentrations, ionic strength, pH, etc.) depending on the actual needs.

(three) kit

In certain embodiments, the Labels in the four compound structures are each different, such as a different fluorophore.

In certain embodiments, kits of the invention further comprise: reagents and/or devices for extracting nucleic acid molecules from a sample; reagents for pretreating nucleic acid molecules; and supports for ligation of nucleic acid molecules to be sequenced a reagent for linking (eg, covalently or non-covalently linking) a nucleic acid molecule to be sequenced to a support; a primer for initial nucleotide polymerization; a polymerase for performing nucleotide polymerization One or more buffer solutions; one or more wash solutions; or any combination thereof.

In certain embodiments, the kits of the invention further comprise reagents and/or devices for extracting nucleic acid molecules from a sample. Methods for extracting nucleic acid molecules from a sample are well known in the art. Therefore, various reagents and/or devices for extracting nucleic acid molecules, such as reagents for disrupting cells, reagents for precipitating DNA, reagents for washing DNA, may be disposed in the kit of the present invention as needed. a reagent for dissolving DNA, a reagent for precipitating RNA, a reagent for washing RNA, a reagent for dissolving RNA, a reagent for removing protein, and a reagent for removing DNA (for example, when the nucleic acid molecule of interest is RNA) An agent for removing RNA (for example, when the nucleic acid molecule of interest is DNA), and any combination thereof.

In certain embodiments, the kit of the invention further comprises an agent for pretreating the nucleic acid molecule. In the kit of the present invention, the reagent for pretreating the nucleic acid molecule is not limited, and may be selected according to actual needs. The reagent for pretreating a nucleic acid molecule includes, for example, a reagent for fragmentation of a nucleic acid molecule (for example, DNase I), a reagent for complementing the end of a nucleic acid molecule (for example, a DNA polymerase such as T4 DNA polymerase, Pfu DNA) a polymerase, Klenow DNA polymerase, a linker molecule, a tag molecule, an agent for linking a linker molecule to a nucleic acid molecule of interest (eg, a ligase, such as T4 DNA ligase), an agent for repairing a nucleic acid nick (eg, loss 3'-5' exonuclease activity but a DNA polymerase showing 5'-3' exonuclease activity), reagents for amplifying nucleic acid molecules (eg, DNA polymerase, primers, dNTPs), for An agent that separates and purifies the nucleic acid molecule (eg, a chromatography column), and any combination thereof.

In certain embodiments, the kit of the invention further comprises a support for ligation of the nucleic acid molecule to be sequenced. The support may have any of the technical features detailed above for the support and any combination thereof.

For example, in the present invention, the support may be made of various suitable materials. Such materials include, for example, inorganics, natural polymers, synthetic polymers, and any combination thereof. Specific examples include, but are not limited to, cellulose, cellulose derivatives (such as nitrocellulose), acrylic resins, glass, silica gel, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide, and Crosslinked polyphenylene bromide such as vinyl benzene (see, for example, Merrifield Biochemistry 1964, 3, 1385-1390), polyacrylamide, latex, dextran, rubber, silicon, plastic, natural sponge, metal plastic, cross-linking dextran (e.g., Sephadex ^TM), agarose gel (Sepharose ^TM), and other supports known to the skilled person.

In certain preferred embodiments, the support for ligation of the nucleic acid molecule to be sequenced may be a solid support comprising an inert substrate or matrix (eg, slides, polymer beads, etc.), said inert substrate or matrix Functionalization has been functionalized, for example, by the use of intermediate materials containing reactive groups that allow covalent attachment of biomolecules such as polynucleotides. Examples of such supports include, but are not limited to, polyacrylamide hydrogels supported on an inert substrate such as glass, in particular polyacrylamide hydrogels as described in WO 2005/065814 and US 2008/0280773, wherein The content of the patent application is hereby incorporated by reference in its entirety. In such embodiments, the biomolecule (eg, a polynucleotide) can be directly covalently attached to an intermediate material (eg, a hydrogel), while the intermediate material itself can be non-covalently attached to the substrate or matrix ( For example, a glass substrate). In certain preferred embodiments, the support is a slide or wafer having a surface modified with a layer of avidin, amino, acrylamide silane or aldehyde based chemical groups.

In the present invention, the support or solid support is not limited by its size, shape and configuration. In some embodiments, the support or solid support is a planar structure, such as a slide, chip, microchip, and/or array. The surface of such a support may be in the form of a planar layer. In some embodiments, the support or surface thereof is non-planar, such as the inner or outer surface of a tube or container. In some embodiments, the support or solid support comprises microspheres or beads. In certain preferred embodiments, the support for ligation of the nucleic acid molecule to be sequenced is an array of beads or wells.

In certain preferred embodiments, the kits of the invention further comprise reagents for attaching (eg, covalently or non-covalently linking) a nucleic acid molecule to be sequenced to a support. Such agents include, for example, agents that activate or modify a nucleic acid molecule (eg, at its 5' end), such as phosphoric acid, a thiol, an amine, a carboxylic acid, or an aldehyde; an agent that activates or modifies the surface of the support, such as an amino group - Alkoxysilane (for example, aminopropyltrimethoxysilane, aminopropyltriethoxysilane, 4-aminobutyltriethoxysilane, etc.); crosslinking agent such as succinic anhydride, phenyl diisosulfide Cyanate (Guo et al., 1994), maleic anhydride (Yang et al., 1998), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) , m-maleimidobenzoic acid-N-hydroxysuccinimide ester (MBS), N-succinimidyl [4-iodoacetyl]aminobenzoic acid (SIAB), 4-(N -maleimidomethyl)cyclohexane-1-carboxylic acid succinimide (SMCC), N-γ-maleimidobutyryloxy-succinimide ester (GMBS), 4-(p-maleimidophenyl)butyric acid succinimide (SMPB); and any combination thereof.

In certain preferred embodiments, the kits of the invention further comprise primers for initiating nucleotide polymerization. In the present invention, the primer is not subject to any limitation as long as it can specifically anneal to a region of the target nucleic acid molecule. In some exemplary embodiments, the primers may be 5-50 bp in length, such as 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40- 45, 45-50bp. In some exemplary embodiments, the primers may comprise naturally occurring or non-naturally occurring nucleotides. In some exemplary embodiments, the primer comprises or consists of a naturally occurring nucleotide. In some exemplary embodiments, the primer comprises a modified nucleotide, such as a locked nucleic acid (LNA). In certain preferred embodiments, the primer comprises a universal primer sequence.

In certain preferred embodiments, the kit of the invention further comprises a polymerase for performing a nucleotide polymerization reaction. In the present invention, polymerization can be carried out using various suitable polymerases. In some exemplary embodiments, the polymerase is capable of synthesizing a new DNA strand (eg, a DNA polymerase) using DNA as a template. In some exemplary embodiments, the polymerase is capable of synthesizing a new DNA strand (eg, a reverse transcriptase) using RNA as a template. In some exemplary embodiments, the polymerase is capable of synthesizing a new RNA strand (eg, RNA polymerase) using DNA or RNA as a template. Accordingly, in certain preferred embodiments, the polymerase is selected from the group consisting of a DNA polymerase, an RNA polymerase, and a reverse transcriptase.

In certain preferred embodiments, the kit of the invention comprises KOD polymerase or a mutant thereof. In certain preferred embodiments, the mutant is selected from the group consisting of KOD POL151, KOD POL157, KOD POL171, KOD POL174, KOD POL376, KOD POL391. In certain preferred embodiments, the kit of the invention comprises KOD POL391, KOD POL171, or a combination thereof.

In certain preferred embodiments, the kit of the invention further comprises a excision reagent capable of cleaving the phosphodiester bond (1) and/or the phosphodiester bond (2) in formula (I') And, does not affect the phosphodiester bond on the duplex backbone.

In certain preferred embodiments, the excision agent is selected from the group consisting of endonuclease IV and alkaline phosphatase.

In certain preferred embodiments, the kit of the invention further comprises one or more buffer solutions. Such buffers include, but are not limited to, buffer solutions for DNase I, buffer solutions for DNA polymerases, buffer solutions for ligases, buffer solutions for eluting nucleic acid molecules, and lysing nucleic acid molecules. A buffer solution, a buffer solution for performing a nucleotide polymerization reaction (for example, PCR), and a buffer solution for performing a ligation reaction. The kit of the present invention may comprise any one or more of the above buffer solutions.

In certain embodiments, the buffer solution for DNA polymerase comprises a monovalent salt ion (eg, sodium ion, chloride ion) and/or a divalent salt ion (eg, magnesium ion, sulfate ion). In certain embodiments, the monovalent salt The concentration of the ion or divalent salt ion in the buffer solution is 1-200 mM, such as 1 mM, 3 mM, 10 mM, 20 mM, 50 mM, 100 mM, 150 mM or 200 mM.

In certain embodiments, the buffer solution for DNA polymerase comprises Tris. In certain embodiments, the concentration of Tris in the buffer solution is from 10 mM to 200 mM, such as 10 mM, 20 mM, 50 mM, 100 mM, 150 mM, or 200 mM.

In certain embodiments, the buffer solution for DNA polymerase comprises an organic solvent such as DMSO or glycerol (glycerol). In certain embodiments, the organic solvent is present in the buffer solution in an amount of from 0.01% to 10%, such as 0.01%, 0.02%, 0.05%, 1%, 2%, 5%, or 10% by mass.

In certain embodiments, the buffer solution for DNA polymerase has a pH of 7.0-9.0, such as 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2. , 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.

In certain embodiments, the buffer solution for DNA polymerase comprises: a monovalent salt ion (eg, sodium ion, chloride ion), a divalent salt ion (eg, magnesium ion, sulfate ion), Tris, and an organic solvent (eg DMSO or glycerol). In certain embodiments, the buffer solution phase has a pH of 7.8.

In certain preferred embodiments, the kit of the invention comprises one or more excision reagents capable of allowing a phosphodiester bond (1) and/or a phosphodiester in a compound of formula (I') The bond (2) is cleaved and does not cleave the phosphodiester bond in the nucleic acid strand backbone to maintain the integrity of the nucleic acid strand backbone.

In certain embodiments, the excision agent is selected from the group consisting of an endonuclease and an IV alkaline phosphatase.

In certain preferred embodiments, the kit of the invention further comprises one or more wash solutions. Examples of such wash solutions include, but are not limited to, phosphate buffer, citrate buffer, Tris-HCl buffer, acetate buffer, carbonate buffer, and the like. The kit of the present invention may comprise any one or more of the above washing solutions.

Modified nucleoside or nucleotide, and the use of the kit

The modified nucleosides or nucleotides of the invention can be used to determine the sequence of a single-stranded polynucleotide of interest.

Accordingly, the present invention also provides the use of a compound as defined in any one of the above, and a kit as defined in any one of the above, for determining the sequence of a single-stranded polynucleotide of interest.

Advantageous effects of the invention

Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

The modified nucleoside or nucleotide provided by the invention has higher stability in sequencing, and the blocking group and the detectable label thereof can be excised under mild conditions without damage to DNA, and Higher resection efficiency can be achieved and even complete resection can be achieved. The modified nucleosides or nucleotides provided by the present invention can be polymerized by a commercially available DNA polymerase and have an acceptable polymerization efficiency, and under suitable conditions, even complete polymerization can be achieved without removing the phosphate group. The ester protecting group can be directly excised, which simplifies the method of excision.

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and embodiments. The various objects and advantageous aspects of the invention will be apparent to those skilled in the <

Detailed ways

The invention is described with reference to the following examples which are intended to illustrate, but not limit the invention. The invention is described by way of example, and is not intended to limit the scope of the invention.

Example 1 Using the base T as an example, a commercially available compound I was used as a starting material to prepare a 3'-OH blocked dTTP having a Cy3 fluorescent group.

(1) Conversion of Compound I-1 to Compound II-1

First, 3 mmol of methanol and 3 mmol of tetrazole were added to 10 mL of anhydrous acetonitrile solvent, and then a solution of 0.88 g (1 mmol) of Compound I-1 dissolved in 5 mL of anhydrous acetonitrile was slowly added, and the reaction was stirred at room temperature for 1 hour. Thereafter, 10 mL of an iodine solution was added, and the solvent of the iodine solution was water/pyridine/tetrahydrofuran (volume ratio 2/20/78), the concentration of iodine was 0.15 M, and 1.5 mmol of iodine was contained, and the mixture was stirred at room temperature for 30 minutes. Thereafter, a 5% sodium sulfite solution was slowly added until the color of the iodine element in the solution disappeared. 100 mL of dichloromethane and 100 mL of a saturated sodium chloride solution were added, the organic phase was extracted, and another 100 mL of dichloromethane was added to the aqueous phase for re-extraction. The organic phases were combined, dried over magnesium sulfate and evaporated to remove solvent. The product can be separated and purified by silica gel column chromatography with a 1:1 ratio of ethyl acetate/n-hexane solvent. The compound II-1 was obtained in a yield: 95% (0.78 g).

Nuclear magnetic and mass spectral data of compound II-1: ¹ H NMR (300 MHz, CDCl ₃ ): δ (ppm) 7.84 (s, 1H), 7.3-7.18 (m, 9H), 6.77 (m, 4H), 6.40 (dd , 1H), 5.05 (s, 1H,), 4.21 (s, 1H), 4.09 (m, 3H), 3.75 (s, 6H), 3.47 (m, 1H), 3.32 (d, 1H), 2.67 (m) , 1H), 2.60 (m, 2H), 2.36 (m, 1H), ³¹ P NMR (163 MHz, CDCl ₃ ): δ (ppm) - 2.48, - 2.66. LC-MS (ESI): m/z 827.2 ( M+H ⁺ ).

(2) Conversion of compound II-1 to compound III-1

0.82 g (1 mmol) of compound II-1 was added to a solution of 3% trichloroacetic acid in dichloromethane (5 mL), stirred at room temperature for 3 hours, concentrated by rotary evaporation to give a solution volume of about 1 mL, and 4:1 on a silica gel column. The ethyl acetate/n-hexane solvent was separated and purified to give Compound III-1, yield: 92% (0.48 g).

Nuclear magnetic and mass spectral data of compound III-1: ¹ H NMR (300 MHz, CDCl 3 ): δ (ppm) 8.24 (s, 1H), 6.26 (dd, 1H), 5.39 - 5.34 (m, 1H), 4.23 - 4.13 ( m, 3H), 4.09 (m, 3H), 3.96 (dd, 1H), 3.89 (dd, 1H), 3.80-3.70 (s, 6H) 2.54 - 2.47 (m, 1H), 2.33 - 2.42 (m, 1H) '), LC-MS (ESI): m/z 525.4 (M+H+).

(3) Conversion of compound III-1 to compound IV-1

Pyridine was added to 52.4 mg (0.1 mmol) of compound III-1, and after several times of drying under high vacuum, a new anhydrous pyridine was added, and 800 μL of 1,4-diox was added to the solid after drying. Hexacyclohexane and 300 μL of anhydrous pyridine were added under argon atmosphere to 1,4-dioxol dissolved in 0.11 mmol (22 mg) of 2-chloro-4H-1,3,2-benzodioxan-4-one. 100 μL of six rings, reacted at room temperature, and stirred for 10 minutes. Add 1.5 times equivalent (1.5mmol, 71.6mg) 300 μL of N-N-dimethylformamide of 100 μL of n-butylammonium pyrophosphate and 100 μL of n-butylamine were stirred at room temperature for 15 minutes. Thereafter, 1 mL of an iodine solution was added, and the solvent was water/pyridine/tetrahydrofuran (2/20/78), the concentration of iodine was 0.15 M, and 0.15 mmol of iodine was contained, and the reaction was stirred at room temperature for 30 minutes. Thereafter, a 5% sodium sulfite solution was slowly added until the color of the iodine element in the solution disappeared. Rotate to remove all solvents, add the remaining solids to 5 mL of water to dissolve, then add 5 mL of 30% ammonia water, stir at room temperature, react for 3 hours, remove all solvents by rotary evaporation, and dissolve the remaining solids with a trace of water, with a ratio of 6:4:1. 4 Dioxane/water/ammonia water was used as a solvent to isolate the product on preparative thin-layer chromatography to give Compound IV-1, yield: 30% (18 mg).

Mass spectral data of compound IV-1: MS (MALDI-): m/z = 614.6 (M - 1).

On high performance liquid chromatography (HPLC), 1:1 acetonitrile/water was used as the mobile phase for the flow phase as shown in Figure 1.

(4) Synthesis of compound V-1

Add 50 mL of DMF to 380 mg (5 mmol) of 2-hydroxyacetic acid, and add 1.8 g (6 mmol) of O-(N-succinimidyl)-N,N,N',N'-tetramethylurea tetrafluoroborate and 1.04 mL of N,N-diisopropylethylamine was stirred at room temperature for 30 minutes. Thereafter, 780 mg of N-ethylenediamine trifluoroacetamide was added, and the mixture was stirred at room temperature for 2 hours. All solvents were removed by rotary evaporation and 100 mL of ethyl acetate was added to dissolve the residual solid. It was extracted with a 0.1 M hydrochloric acid solution (2×100 mL), and then extracted with a saturated sodium carbonate solution (2×150 mL), then dried over magnesium sulfate, filtered and evaporated to remove solvent to yield compound V-1, yield: 75% (0.8g).

Nuclear magnetic and mass spectral data of the compound V-1: ¹ H NMR (300 MHz, CDCl 3 ): δ (ppm) 4.05 (s, 2H), 3.40 - 3.30 (m, 4H), LC-MS (ESI): m/z 215.2 (M+H+).

(5) Conversion of compound V-1 to synthetic compound VI-1

214 mg (1 mmol) of compound V-1 was added to 10 mL of anhydrous dichloromethane, and 205 μL of N,N-diisopropylethylamine was added, followed by the addition of 283 mg (1.2 mmol) of 2-cyanoethyl at 0 °C. 2 mL of anhydrous dichloromethane of N,N-diisopropylchlorophosphoramidite was further stirred at 0 °C for 10 minutes. The temperature was slowly raised to room temperature and reacted for 2 hours. Thereafter, 20 mL of dichloromethane and 50 mL of a saturated sodium hydrogencarbonate solution were added, and after extraction, magnesium sulfate was added to dryness to remove the solvent. Compound VI-1 was isolated on a silica gel column using 1:1 ethyl acetate / n-hexane as eluent. Yield: 85% (352 mg).

Nuclear magnetic and mass spectral data of compound VI-1: ¹ H NMR (300 MHz, CDCl 3 ): δ (ppm) 4.16 (s, 2H), 3.8 (m, 2H), 3.40 - 3.30 (m, 6H), 3.10 (t, 2H), 1.16 (d, 12H) LC-MS (ESI): m/z 415.3 (M+H+).

(6) Conversion of compound VI-1 to compound VII-1

207 mg (0.5 mmol) of compound VI-1 was dissolved in 3 mL of anhydrous acetonitrile, and the solution was slowly added to a solution of 70 mg (1 mmol) of tetrazole and 239 mg (1 mmol) of 2-nitro-5-hydroxy-benzoic acid. The butyl ester in anhydrous acetonitrile (7 mL) was stirred at room temperature for 30 minutes. Then, 5 mL of an iodine solution was added, and the solvent was water/pyridine/tetrahydrofuran (2/20/78), the concentration of iodine was 0.15 M, and 0.75 mmol of iodine was contained, and the mixture was stirred at room temperature for 30 minutes. Thereafter, a 5% sodium sulfite solution was slowly added until the color of the iodine element in the solution disappeared. All solvents were removed by rotary evaporation, 5 mL of water was added to dissolve the remaining solid, and 5 mL of 2M potassium hydroxide was added and stirred at room temperature for 2 hours. The pH of the solution was adjusted to be acidic, and a large amount of methanol was used to dissolve the solid, followed by rotary evaporation to remove the solvent. Recrystallization of the remaining solid using water as a solvent gave Compound VII-1, yield: 36% (131 mg).

Nuclear magnetic and mass spectral data of compound VII-1: ¹ H NMR (300 MHz, CDCl 3 ): δ (ppm) 8.23 - 8.11 (m, 2H), 7.64 (m, 1H), 4.45 (s, 2H), 3.40 - 3.30 ( t, 2H), 2.90 (t, 2H), LC-MS (ESI): m/z 362.1 (MH-).

(7) Fluorescent labeling of compound VII-1 to obtain compound VIII-1

12.8 mg (20 umol) of Cy3 N-hydroxysuccinimide ester was dissolved in 1 mL of DMF, and 4.2 μL (24 μmol) of N,N-diisopropylethylamine and 8.7 mg (24 μmol) of compound dissolved therein were added thereto. DMF of VII-1 (1 mL), stirred at room temperature, reacted for 2 hours, then evaporated to remove solvent. The remaining solid was dissolved by adding as little DMF as possible, and then purified by HPLC using 100% acetonitrile as a mobile phase to give Compound VIII-1, yield: 95% (18.6 mg).

Mass spectral data for compound VIII-1: LC-MS (ESI): m/z 975.2 (M-H-).

(8) Compound IV-1 is linked to compound VIII-1 to form dTTP having a fluorescent group Cy3 and 3'-OH is reversibly blocked.

2 μL (12 μmol) of N,N-diisopropylethylamine and 3.6 mg (12 μmol) of tetrafluoroboric acid O-(N-succinyl) were added to 1 mL of DMF in which 9.8 mg (10 μmol) of Compound VIII-1 was dissolved. Amino)-N,N,N',N'-tetramethylurea, after reacting for 30 minutes, add 1 mL of 0.1 M sodium hydrogen carbonate solution dissolved in 6.2 mg (10 μmol) of compound IV-1, and stir at room temperature. 2 hours. Remove all solvents in high vacuum, add as little water as possible, then use 1:1 water/acetonitrile and 0.05 M triethylamine solution (pH 7.0) as the mobile phase, and purify on HPLC to obtain the label labeled Cy3 and 3. '-OH blocked dTTP. Yield: 90% (14.1 mg).

Mass spectral data of the final product: LC-MS (ESI): m/z 1573.2 (M-H-);

Example 2, taking base A as an example, prepares a 3'-OH blocked nucleotide (dATP) having a Cy3 fluorescent group.

An intermediate (Compound IV-2) for the synthesis of dATP was prepared by referring to the procedures of Examples 1 (1) to (3). Compound IV-2 The structure is as follows:

Mass spectral data for compound IV-2: MS (MALDI -): m/z = 636.1 (M -1).

On high performance liquid chromatography, 1:1 acetonitrile/water was used as the mobile phase, and the effluent time was as shown in Fig. 3.

Referring to the procedure of Example 1, 3'-OH blocked dATP having a Cy3 fluorescent group was prepared.

Example 3 Polymerization of Modified dTTP

In this example, the modified dTTP of the present invention was polymerized using an exemplary template and primers, and the polymerization efficiency was tested to examine the effect of the polymerase and polymerization conditions on the polymerization efficiency.

(1) Template and primer sequences

Template: 5'-CAACAGAAGGATTCTGGCGAACCGGAGGCTGAA--3' (SEQ ID NO: 1)

Primer: 3'-TGTCTTCCTAAGACCGCTTGGCCTCCGACTT-5' (SEQ ID NO: 2)

(2) polymerase

Theminator (NEB), Taq (NEB), BST 2.0 (NEB), BST 3.0 (NEB), 9°Nm (NEB), KOD (merckmillipore)

Mutants of KOD polymerase: KOD POL151, KOD POL157, KOD POL171, KOD POL174, KOD POL376, KOD POL391

The above polymerase or a mutant thereof is obtained by purchase.

(3) Sequencing system

BGISEQ-500 Sequencing System (refer to the BGISEQ-500 User Manual for the method of operation)

(4) Test process

Figure 4 exemplifies the testing process.

The chip used for the test consisted of two polymerization reaction zones: zone 1 and zone 2, wherein zone 1 was the reaction of the reference group and zone 2 was the reaction of the group to be tested. It is known that Cy3-modified dideoxy dTTP can be polymerized 100% by Taq DNA polymerase under the test conditions mentioned below, thus serving as a reference group.

Cy3 modified dideoxy dTTP (triethylamine salt) was purchased from Jena Bioscience and its chemical structure is as follows:

Load the chip. First, the chip is photographed, and the background signal value is collected. Then, the reaction solution 1 containing Taq DNA polymerase and Cy3 modified dideoxy dTTP is added to the region 1, and the reaction solution 2 is added to the region 2, which contains the test to be tested. The polymerase and the reversibly blocked dTTP with the fluorophore Cy3 prepared in Example 1 were each polymerized at a suitable temperature for 10 minutes, and the concentration of dTTP was 10 μM. The buffer was polymerase product specification. Recommended buffer system. After the reaction, the chips were repeatedly washed with 1X phosphate buffer to remove unreacted dTTP; 1X phosphate buffer containing 10 mM of vitamin C was pumped, and both sides of the chip were photographed to collect signal values.

Each test yielded four values, including the blank background values (a and b) of the reference group and the test group, and the signal values (A and B) of the reference group and the test group after polymerization. It is known that the polymerization efficiency of the control group is 100%, and the polymerization efficiency of the test group can be calculated by the following equation:

The polymerization efficiency of the test group = (B-b) / (A - a) * 100%.

Polymerization was carried out using different polymerases, and the test results are shown in Table 1 (the difference between the background values of the different batch tests and the signals of the reference group).

Table 1

The test results showed that the polymerases in Table 1 were able to polymerize the dTTP prepared in Example 1, but the polymerization efficiency of other polymerases was lower than that of KOD. The polymerization efficiency using KOD as a polymerase is acceptable.

Further, mutants KOD POL151, KOD POL157, KOD POL171, using KOD polymerase, KOD POL174, KOD POL376, and KOD POL391 were tested as polymerases. These mutants have a poor polymerization effect when polymerizing some reversible blocking dNTPs in the prior art.

The dideoxy dTTP modified with Taq DNA polymerase and Cy3 was used as a reference group, and the template shown in SEQ ID NO: 1 and the primer shown in SEQ ID NO: 2, and the same experimental method as above, were used for polymerization for 10 minutes. . The test results are shown in Table 2.

Table 2

As can be seen from Table 2, the polymerases KOD POL391 and KOD POL171 had higher polymerization efficiencies for the dTTP prepared in Example 1.

Further, the inventors optimized the reaction conditions (including the pH of the reaction solution, the salt concentration, the buffer system concentration, the additive, and the reaction temperature) for polymerization using KOD POL391.

The initial reaction conditions were as follows: the pH of the reaction solution was 9.0, and contained 50 mM sodium chloride, 1 mM magnesium sulfate, 50 mM Tris, 0.05% Tween-20, and the reaction temperature was 55 °C. When the reaction conditions were optimized, the reference group was not used, but a comparison test was performed on the two reaction areas of the chip, and the polymerization time was 5 minutes. The better conditions that were first optimized were used in subsequent tests. The test results are shown in Table 3-1 to Table 3-6.

Table 3-1

pHpH	pH 9.0pH 9.0	pH 8.5pH 8.5	pH 8.0pH 8.0	pH7.8pH 7.8	pH 7.5pH 7.5	pH 7.0pH 7.0
聚合效率Polymerization efficiency	55.1255.12	76.6576.65	89.9089.90	93.7693.76	80.2180.21	37.3837.38

Other conditions: The reaction solution contained 50 mM sodium chloride, 1 mM magnesium sulfate, 50 mM Tris, 0.05% Tween-20, and the reaction temperature was 55 °C.

Table 3-2

氯化钠浓度Sodium chloride concentration	20mM20mM	100mM100mM
聚合效率Polymerization efficiency	96.3396.33	92.5092.50

Other conditions: The reaction solution was pH 7.8, and contained 1 mM magnesium sulfate, 50 mM Tris, 0.05% Tween-20, and the reaction temperature was 55 °C.

Table 3-3

硫酸镁浓度Magnesium sulfate concentration	10mM10mM	3mM3mM
聚合效率Polymerization efficiency	93.9493.94	97.4097.40

Other conditions: The reaction solution was pH 7.8, and contained 20 mM sodium chloride, 50 mM Tris, 0.05% Tween-20, and the reaction temperature was 55 °C.

Table 3-4

Tris浓度Tris concentration	100mM100mM	20mM20mM
聚合效率Polymerization efficiency	94.6794.67	93.2893.28

Other conditions: The reaction solution was pH 7.8, and contained 20 mM sodium chloride, 3 mM magnesium sulfate, 0.05% Tween-20, and the reaction temperature was 55 °C.

Table 3-5

添加剂additive	2％DMSO2% DMSO	5％DMSO5% DMSO	5％甘油5% glycerin
聚合效率Polymerization efficiency	98.4598.45	99.1099.10	88.9988.99

Other conditions: The reaction solution was pH 7.8, and contained 20 mM sodium chloride, 3 mM magnesium sulfate, 50 mM Tris, 0.05% Tween-20, and the reaction temperature was 55 °C.

Table 3-6

反应温度temperature reflex	57℃57°C	60℃60 ° C	63℃63°C
聚合效率Polymerization efficiency	99.0299.02	98.4398.43	93.7793.77

Other conditions: The reaction solution was pH 7.8 and contained 20 mM sodium chloride, 3 mM magnesium sulfate, 50 mM Tris, 0.05% Tween-20, 5% DMSO.

It can be seen from Table 3-1 to Table 3-6 that changing the pH to 7.8 and adding 5% DMSO have the greatest influence on the polymerization efficiency, and other conditions have almost no positive effect on the pH. The reaction conditions finally optimized were: pH 7.8 of the reaction solution, containing 20 mM sodium chloride, 3 mM magnesium sulfate, 50 mM Tris, 5% DMSO, 0.05% Tween-20, and the reaction temperature was 57 °C.

The polymerization efficiency of the polymerase KOD POL391 and KOD POL171 against the dTTP obtained in Example 1 was measured under the conditions of the final optimization, and the polymerization reaction was carried out for 10 minutes. The reaction of the Cy3-modified dideoxy dTTP polymerized by Taq DNA polymerase was used as a reference group. The results are shown in Table 4.

Table 4

As shown in Table 4, the Cy3-modified reversibly blocked dTTP prepared in Example 1 can be efficiently polymerized by KOD POL391 or KOD POL171, and the polymerization efficiency is close to 100%.

Example 4 Removal of Blocking Groups

In this example, the dTTP which is reversibly blocked but does not have a fluorescent group is first polymerized. The dTTP which was used in the present embodiment to be reversibly blocked without a fluorescent group was the compound IV-1 in Example 1, and the structure was as follows. Under optimized conditions, it can be 100% polymerized.

After the polymerization, the blocking group was excised using endonuclease IV (NEB), and the efficiency of excision was tested by the addition of the next base after excision.

Figure 5 exemplifies the testing process.

First, a chip having two reaction regions (region 1 and region 2) is loaded, wherein the reaction of the reference group is performed on the region 1, and the reaction of the group to be tested is performed on the region 2. Two different primers are added to the two regions, wherein the primer of the reference group is as shown in SEQ ID NO: 3, which is one more T at the 3 end than the primer of the test group (as shown in SEQ ID NO: 2). Base, therefore, after adding a T base to the test group and excising, the reference group and the test composition are systems having the same sequence, and the excision efficiency of the test group can be determined by comparing the signals of the next base.

In Region 1 and Region 2, polymerase KOD POL391 was used, and under the finally optimized reaction conditions obtained in Example 3, Compound IV-1 was polymerized, the concentration of Compound IV was 10 μM, and polymerization was carried out for 10 minutes. The unreacted reagent was washed away, and the excision reagent 4 (100 U/mL) was used as the excision reagent, and the excision reaction was carried out in NEB buffer 3 at a reaction temperature of 37 ° C, and the excision time was 10 min, 20 min, 40 min or 60 min. The chip is washed, and then a signal is acquired on the chip to obtain background values a and b of the reference group and the group to be tested. Taq DNA and Cy3 modified ddGTP were added, and polymerization was carried out for 10 minutes, after which unreacted ddGTP was washed away, and a signal was collected to obtain signal values A and B of the reference group and the test group. The cutting efficiency can be calculated using the equation below.

Excision efficiency == (B-b) / (A-a) * 100%.

Table 5 and Figure 6 show the efficiency of the ablation at different ablation times.

table 5

As can be seen from Table 5 and Figure 6, the cutting efficiency entered the plateau after 20 minutes, and the final cutting efficiency reached 100%.

Example 5 Removal of Fluorescent Groups

In this example, the fluorophore was removed by cleaving the phosphodiester bond (2) in the modified nucleotide of the present invention, and the efficiency of excision was tested.

Figure 7 exemplifies the testing process.

The CyT-modified and reversibly blocked dTTP prepared in Example 1 was polymerized on a chip, and the polymerase was KOD POL391. The reaction conditions were the final optimized reaction conditions of Example 1, and the concentration of dTTP was 10 μM. The signal values are collected before and after the polymerization, wherein the signal value before the polymerization is the background A1, and the signal value after the polymerization is a, and then the alkaline phosphatase (NEB, item number M0371S) is used for excision, and the 2, 5, 10 or 20 are respectively cut off. Minutes, after the excision, wash off the reagents, and then collect the signal value, which is A2, and calculate the resection efficiency according to the following equation:

Excision efficiency = (a-A2) / (a-A1) * 100%.

The test results are shown in Table 6 and Figure 8.

Table 6

切除时间(min)Cutting time (min)	A1A1	aa	A2A2	切除效率Resection efficiency
22	434434	60126012	13821382	83％83%
55	333333	45674567	672672	92％92%
1010	282282	47184718	370370	98％98%
2020	426426	58405840	412412	100％100%

As can be seen from Table 6 and Figure 8, the fluorophore on the nucleic acid strand can be completely removed after 20 min of excision.

Example 6 tested the stability of the azide methylene blocking group and the stability of the methyl phosphate blocking group.

Test compound:

(1) dTTP having a fluorescent group Cy3 and 3'-OH blocked by methyl phosphate prepared in Example 1;

(2) dTTP having a fluorescent group Cy3 and 3'-OH blocked by an azide methylene group (structure is as follows, purchased by mychemlab).

Testing process:

1 M tris-buffer (pH 8.0) (0.3 mL) and human serum albumin were added to 1 mg of the two compound samples, respectively, and stirred at 65 ° C for 36 hours; then, using liquid chromatography-mass spectrometry (LC) for two One sample was analyzed (LC-MS), and a 1:1 acetonitrile:acetic acid triethylamine salt aqueous solution (0.05 M, pH 7) was used as a mobile phase to detect whether two samples had a 3' end protecting group removed. Molecular mass peaks of molecular bases or fluorescent modified groups are removed.

The LC results of dTTP blocked by methyl phosphate are shown in Figure 9. A very small new peak was generated on the LC map and identified by mass spectrometry with a molecular weight of 780.1 (MH) ^- and the structure is as follows.

The LC results of dTTP blocked by azide methylene group are shown in Figure 10. There are two larger new peaks and one smaller new peak on the LC map. Identification by mass spectrometry, structure and molecular weight (from left to right in the figure):

A nucleotide with a protecting group at the 3' end and a fluorescent modification at the base, 1482.3 (MH) ^- .

While the invention has been described in detail, the embodiments of the invention . The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

a compound having the structure represented by the formula (I),

Wherein R 1 and R 3 are each independently selected from

A is selected from the group consisting of phenyl, naphthyl, anthryl and pyridyl;

R 2 is selected from the group consisting of a nitro group, a halogenated C 1-4 alkyl group, a halogen, a hydrogen, an aldehyde group,
Wherein Q is independently selected from C 1-4 alkyl;

R 4 is selected from -H, a monophosphate group (-PO 3 H 2 ), a diphosphate group (-PO 3 H-PO 3 H 2 ), a triphosphate group (-PO 3 H-PO 3 H-PO 3 H 2 ) and a tetraphosphoric acid group (-PO 3 H-PO 3 H-PO 3 H-PO 3 H 2 );

R 6 is hydrogen or a hydroxyl group;

m and n are each independently selected from 0, 1, 2, 3, 4, 5;

L is a linking group or does not exist;

Base represents a base, such as a purine base or a pyrimidine base, for example, one selected from the group consisting of A, T, U, C, and G;

Label indicates a detectable label, such as a fluorophore;

Blocker indicates a blocking group.
The compound of claim 1 wherein R 1 is selected from
Preferably, R 1 is
The compound of claim 1 or 2, wherein R 2 is selected from the group consisting of nitro, trifluoromethyl, fluoro, chloro, hydrogen and aldehyde; preferably, R 2 is nitro.
The compound according to any one of claims 1 to 3, wherein R 3 is selected from
Preferably, R 3 is
A compound according to any one of claims 1 to 4, wherein
From:

Preferably,
for
A compound according to any one of claims 1 to 5, wherein R 4 is a triphosphate group (-PO 3 H-PO 3 H-PO 3 H 2 ).
A compound according to any one of claims 1 to 6, wherein R 6 is hydrogen.
The compound according to any one of claims 1 to 7, wherein the Label is selected from the group consisting of Cy3, Cy3.5, Cy5 and Cy5.5.
A compound according to any one of claims 1-8, wherein m is 1.
The compound of any one of claims 1-9, wherein n is 1.
A compound according to any one of claims 1 to 10, wherein L is
The compound of any one of claims 1-11, wherein the structure of the Blocker is:

Wherein, Ra 1 and Ra 2 are each independently selected from the group consisting of H, F, -CF 3 , -CHF 2 , -CH 2 F, -CH 2 W, -COOW, -CONHW;

W is selected from a C 1 -C 6 alkyl group.
The compound of any one of claims 1-11, wherein the structure of the Blocker is:

Wherein Rb 1 , Rb 2 , Rb 3 , Rb 4 and Rb 5 are each independently selected from H and C 1 -C 6 alkyl.
The compound of any one of claims 1-11, wherein the structure of the Blocker is:

Wherein Rc 1 and Rc 2 are each independently selected from the group consisting of H, F, Cl and -CF 3 .
A compound according to any one of claims 1 to 11, which has the structure represented by the formula (I'):

Wherein R 5 is selected from C 1-4 alkyl;

Preferably, R 5 is methyl or ethyl.
A compound according to any one of claims 1 to 15, which has the structure represented by the formula (II)

Preferably, R 2 is a nitro group;

Preferably, the Label is Cy3.
A compound according to any one of claims 1 to 16 which is selected from the group consisting of:
A method of preparing a growing polynucleotide that is complementary to a target single-stranded polynucleotide in a sequencing reaction, comprising incorporating the compound of any one of claims 1-17 into the growing complementary polynucleotide, wherein Incorporation of the compounds prevents any subsequent nucleotides from being introduced into the growing complementary polynucleotide;

Preferably, the incorporation of the compound is achieved by a terminal transferase, a terminal polymerase or a reverse transcriptase;

Preferably, the method comprises: incorporating the compound into a growing complementary polynucleotide using a polymerase;

Preferably, the method comprises performing a nucleotide polymerization using a polymerase under conditions allowing the polymerase to undergo nucleotide polymerization, thereby incorporating the compound into the 3' end of the growing complementary polynucleotide.
A method of determining the sequence of a single-stranded polynucleotide of interest, comprising: monitoring the sequential incorporation of a complementary nucleotide, wherein the at least one complementary nucleotide incorporated is a compound of any of claims 1-17, and Detecting a detectable label carried by the compound;

Preferably, the blocking group and the detectable label in the compound are removed prior to introduction of the next complementary nucleotide;

Preferably, the blocking group and the detectable label are simultaneously removed;

Preferably, the blocking group and the detectable label are removed in succession; for example, the blocking group is removed before or after the detectable label is removed.
The method of claim 19 comprising the steps of:

(a) providing a mixture comprising a duplex, a compound according to any one of claims 1-17, a polymerase and a excision reagent; the duplex comprising a growing nucleic acid strand and a nucleic acid molecule to be sequenced;

(b) Carry out a reaction cycle comprising the following steps (i), (ii) and (iii):

Step (i): using a polymerase to incorporate the compound into a growing nucleic acid strand to form a nucleic acid intermediate comprising a blocking group and a detectable label;

Step (ii): detecting a detectable label on the nucleic acid intermediate;

Step (iii): removing the blocking group on the nucleic acid intermediate using a resection reagent;

Preferably, the reaction cycle further comprises the step (iv): removing the detectable label on the nucleic acid intermediate using a scavenging reagent;

Preferably, the ablation reagent used in the step (iii) and the step (iv) is the same reagent;

Preferably, the ablation reagents used in step (iii) and step (iv) are different reagents.
The method of claim 19, wherein the at least one complementary nucleotide incorporated is a compound of the formula (I'), the method comprising the steps of:

(1) providing a duplex comprising a growing nucleic acid strand and a nucleic acid molecule to be sequenced, the duplex being linked to a support;

(2) adding a polymerase for performing nucleotide polymerization, and first, second, third, and fourth compounds, thereby forming a reaction system containing a solution phase and a solid phase; wherein the four compounds are respectively Derivatives of nucleotides A, (T/U), C and G, each having the structure represented by the general formula (I) and having a base complementary pairing ability;

(3) performing nucleotide polymerization using a polymerase under conditions allowing the polymerase to undergo nucleotide polymerization, thereby incorporating one of the four compounds into the 3' end of the growing nucleic acid strand;

(4) removing the solution phase of the reaction system of the previous step, leaving the duplex attached to the support, and detecting the signal emitted by the detectable label on the duplex or the growing nucleic acid strand;

(5) adding a scavenging reagent such that the duplex or the growing nucleic acid strand is contacted with a scavenging reagent in a reaction system containing a solution phase and a solid phase; wherein the excision reagent enables the incorporation of the growing nucleic acid strand The phosphodiester bond (1) and/or the phosphodiester bond (2) in the compound at the 3' end are cleaved and do not affect the phosphodiester bond on the duplex backbone;

(6) removing the solution phase of the reaction system of the previous step;

Preferably, the method further comprises the steps of:

(7) repeating steps (2)-(6) or steps (2)-(4) one or more times;

Optionally, the washing operation is performed after any of the steps including the removing operation.
The method of claim 21, wherein the duplex in step (1) is obtained by a method comprising the steps of:

Primers are provided to anneal the primer to the nucleic acid molecule to be sequenced, the primer being the starting nucleic acid strand for growth, A duplex attached to the support is formed with the nucleic acid molecule to be sequenced.
The method of claim 21 or 22, wherein the polymerase in the step (3) is selected from the group consisting of KOD polymerase or a mutant thereof (for example, KOD POL151, KOD POL157, KOD POL171, KOD POL174, KOD POL376, KOD POL391);

Preferably, the polymerase is selected from the group consisting of KOD POL391 and KOD POL171.
The method of any one of claims 21 to 23, wherein the solution phase in the step (2) comprises a monovalent salt ion (e.g., sodium ion, chloride ion) and/or a divalent salt ion (e.g., magnesium ion, sulfate ion) ;

Preferably, the concentration of the monovalent salt ion or divalent salt ion in the solution phase is 1-200 mM;

Preferably, the solution phase in the step (2) comprises a buffer solution, such as a Tris buffer solution;

Preferably, the concentration of Tris in the solution phase is from 10 mM to 200 mM;

Preferably, the solution phase in the step (2) comprises an organic solvent such as DMSO or glycerol;

Preferably, the organic solvent has a mass content in the solution phase of 0.01% to 10%;

Preferably, the pH of the solution phase in the step (2) is 7.0-9.0;

Preferably, the polymerization in the step (3) is carried out at 50 to 65 °C.
The method according to any one of claims 21 to 24, further comprising, after step (4), based on the principle of base complementary pairing, according to the type of compound incorporated in the 3' end of the growing nucleic acid strand in step (3) Determining the type of base at the corresponding position in the nucleic acid molecule to be sequenced.
The method according to any one of claims 21 to 25, wherein in the step (5), the excision of the blocking group and the excision of the detectable label are performed simultaneously, or the excision of the blocking group and the detection of the detectable label Excision step by step;

Preferably, the step (5) comprises:

Step (5-1): adding a first excision reagent, contacting the duplex or the grown nucleic acid strand with the first excision reagent in a reaction system containing a solution phase and a solid phase without affecting the duplex a phosphodiester bond (1) in a compound incorporated at the 3' end of the growing nucleic acid strand under conditions of a phosphodiester bond on the backbone;

Step (5-2): adding a second excision reagent, so that the duplex or the grown nucleic acid strand is contacted with the first excision reagent in a reaction system containing a solution phase and a solid phase without affecting the duplex a phosphodiester bond (2) in a compound incorporated at the 3' end of the growing nucleic acid strand under conditions of a phosphodiester bond on the backbone;

Preferably, the first excision reagent is endonuclease IV;

Preferably, the second excision reagent is alkaline phosphatase.
A kit comprising first, second, third and fourth compounds, each of said first, second, third and fourth compounds being a compound according to any one of claims 1-17, said four The compounds are derivatives of nucleotides A, (T/U), C and G, respectively, and have a base complementary pairing ability;

Preferably, the Labels in the four compound structures are different, for example, different fluorophores.
The kit of claim 27, the kit comprising a polymerase for performing a nucleotide polymerization reaction;

Preferably, the kit comprises KOD polymerase or a mutant thereof;

Preferably, the mutant is selected from one or more of KOD POL151, KOD POL157, KOD POL171, KOD POL174, KOD POL376, KOD POL391;

Preferably, the kit comprises KOD POL391, KOD POL171 or a combination thereof.
The kit according to claim 27 or 29, further comprising a excision reagent capable of cleavage of a phosphodiester bond (1) and/or a phosphodiester bond (2) in the formula (I'), Moreover, it does not affect the phosphodiester bond on the duplex backbone;

Preferably, the excision agent is selected from the group consisting of endonuclease IV and alkaline phosphatase.
The kit according to any one of claims 27 to 29, further comprising: reagents and/or means for extracting nucleic acid molecules from the sample; reagents for pretreating the nucleic acid molecules; and for connecting nucleic acids to be sequenced a support for a molecule; an agent for linking (eg, covalently or non-covalently linking) a nucleic acid molecule to be sequenced to a support; a primer for initial nucleotide polymerization; for performing nucleotide polymerization The reacted polymerase; one or more buffer solutions; one or more wash solutions; or any combination thereof.
A kit according to any one of claims 27 to 30, the kit comprising a buffer solution;

Preferably, the buffer solution comprises monovalent salt ions (such as sodium ions, chloride ions) and/or divalent salt ions (such as magnesium ions, sulfate ions);

Preferably, the concentration of the monovalent salt ion or divalent salt ion in the buffer solution is 1-200 mM;

Preferably, the buffer solution comprises Tris;

Preferably, the concentration of Tris in the buffer solution is 10 mM to 200 mM;

Preferably, the pH of the buffer solution is 7.0-9.0;

Preferably, the buffer solution comprises an organic solvent such as DMSO or glycerol.
Use of a compound according to any one of claims 1-17, or a kit according to any one of claims 27-31, for determining the sequence of a single-stranded polynucleotide of interest.