WO2024164823A1 - Nucleotide analogue and use thereof in sequencing - Google Patents

Abstract

The present invention provides a compound having a structural formula as shown in formula (I) and a use thereof in sequencing. The compound introduces an orthogonal cleavage group on nucleotide, such that when the compound is used as an alternative incorporation primer of an invertible terminator for extension, the orthogonal cleavage group serving as a protective group can be quickly cut off by means of an inverse electron demand Diels-Alder (IEDDA) reaction, so as to expose 3'-OH for a biochemical reaction of the next round, thereby greatly shortening the reaction time in a sequencing process, reducing a phasing value, finally reducing the error rate in the sequencing process, and improving the sequencing accuracy.

Description

Nucleotide analogs and their applications in sequencing

The present invention claims the priority of Chinese patent application filed with the Chinese Patent Office on February 7, 2023, with application number 202310076009X and application name “Nucleotide analogs and their applications in sequencing”, the entire contents of which are incorporated by reference into the present invention.

Technical Field

The present application relates to the field of nucleotide technology, and in particular to nucleotide analogs and their application in sequencing.

Background Art

Next-generation sequencing (NGS), also known as high-throughput sequencing, is a DNA sequencing technology developed based on PCR and gene chips. The second-generation sequencing pioneered the introduction of reversible terminators, thereby realizing sequencing by synthesis. Its basic principle is to determine the sequence of DNA by capturing the special markers (generally fluorescent molecular markers) carried by the newly added bases during DNA replication. In order to detect only one base per cycle, the base addition process requires the use of nucleotide analogs containing blocking groups that carry markers as reversible terminators, so as to react them with the target nucleic acid and DNA polymerase for biochemical reactions. The target nucleic acid is used as a template to extend the primer and incorporate the nucleotide analogs. The nucleotide analogs with blocking groups will terminate the next extension of the primer until the deblocking reagent is added, and the next biochemical reaction can be carried out. This cycle is repeated many times, and the special markers carried by it are detected in each cycle, and the sequence of the target nucleic acid is analyzed. For example, the nucleotide analog obtained by introducing an azidomethyl group as a blocking group to protect the 3′-OH of the nucleotide will block the incorporation of the next base during the extension process of the target nucleic acid. After optical detection and analysis, a trivalent phosphorus reagent is added to remove the azidomethyl group, and then the next round of biochemical reaction is carried out. This cycle is repeated many times, and detection is performed in each cycle to obtain the nucleic acid sequence.

In the process of second-generation sequencing, phasing refers to the process of sequencing while synthesizing, in which a part of the DNA chain cannot be completely incorporated into the cluster by the polymerase in the next round of sequencing cycle due to incomplete removal of the 3′ terminator and the fluorophore. Pre-phasing, on the other hand, refers to the incorporation of nucleotide analogs in the absence of an effective 3′ terminator, and the incorporation process is performed in advance for one cycle. Taking the fluorescently labeled nucleotides protected by azidomethyl as an example, sodium azide is required during the synthesis of the azide group, and sodium azide is explosive. At the same time, as an organic azide compound, its thermal stability is poor at the sequencing biochemical reaction temperature, and the azide methyl group is easily detached to expose the 3′-OH, causing the next round of biochemical reaction in sequencing, resulting in a high Pre-phasing value. In addition, organic azide compounds are easily affected by reducing agents during storage and transfer; and the removal of the azide methyl group requires the use of reducing agents such as easily oxidized trivalent phosphorus reagents. In addition, when using excision reagents, the excision time and excision efficiency affect the next biochemical reaction. Incomplete excision will lead to too high phasing values, and too long excision reaction time will lead to Too low efficiency will affect the biochemical reaction time of sequencing, increase sequencing costs, etc. Therefore, it is necessary to provide a nucleotide analog for sequencing, which can reduce the reaction time and phasing value in the sequencing process, and ultimately reduce the error rate and improve the accuracy.

Summary of the invention

The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes a nucleotide analog and its application in sequencing, which can be quickly removed as a replacement for the reversible terminator in sequencing to expose 3′-OH, reduce the reaction time in the sequencing process, reduce the phasing value in the sequencing process, and ultimately reduce the error rate and improve the accuracy.

In the first aspect of the present application, a nucleotide analogue having a structural formula as shown in formula (I) is provided:

Wherein, R ¹ includes an optional orthogonal breaking group;

R ² includes a base;

R ³ includes any one of a hydroxyl group and a phosphate group;

R ⁴ includes any one of hydrogen and OR ⁵ , and R ⁵ is an optional orthogonal breaking group;

At least one of R ¹ , R ² , R ³ , and R ⁴ is also linked to a detectable label.

The compounds according to the embodiments of the present application have at least the following beneficial effects:

The compounds provided in the examples of the present application introduce an orthogonal cleavage group on the nucleotide. When such a compound is incorporated into a primer as a replacement for a reversible terminator for extension, the orthogonal cleavage group as a protecting group can be quickly removed through an inverse electron demand Diels-Alder (IEDDA) reaction, thereby exposing 3′-OH for the next round of biochemical reaction, greatly shortening the reaction time in the sequencing process, reducing the phasing value, and ultimately reducing the error rate in the sequencing process and improving the accuracy of sequencing.

The orthogonal cleavage group refers to a group based on the bioorthogonal cleavage (click to release) principle, which has bioorthogonality and controllable bond cleavage properties.

In some embodiments of the present application, the orthogonal cleavage group is a dienophile group. By using the dienophile group as a linker for orthogonal cleavage, an inverse electron demand Diels-Alder (IEDDA) click reaction occurs, so that the orthogonal cleavage group as a protecting group can be quickly released from the 3′-OH position, achieving a shorter excision time and higher excision efficiency, and reducing the time required for sequencing.

In some embodiments of the present application, the orthogonal breaking group is -LR ⁶ ;

Wherein, L is selected from non-existent, substituted or unsubstituted C1-C10 alkylene, substituted or unsubstituted C1-C10 heteroalkylene any one of substituted or unsubstituted C1-C10 cycloalkylene, substituted or unsubstituted C1-C10 heterocycloalkylene, substituted or unsubstituted C6-C10 arylene and substituted or unsubstituted C6-C10 heteroarylene;

^R6 is selected from a substituted or unsubstituted trans-cyclooctene group, a substituted or unsubstituted norbornene group, a substituted or unsubstituted benzonorbornene group, a substituted or unsubstituted vinyl group, a substituted or unsubstituted cyano group, and a substituted or unsubstituted azidophenyl group.

In some embodiments of the present application, the substituent of the group in the above L or ^R6 can be selected from at least one of halogen, haloalkyl, cyano, amino, nitro, carboxyl, thiol, sulfonic acid, acyl, amide, sulfonyl, carbonyl, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted alkoxy, unsubstituted heterocycloalkyl, unsubstituted aryl, and unsubstituted heteroaryl.

The heteroatoms in the heteroalkyl, heteroalkylene, heteroaryl, heteroarylene, heterocycloalkyl, and heterocycloalkylene include but are not limited to atoms such as O, N, P, S, and Si, and the number of heteroatoms can be at least one.

In some embodiments of the present application, L is selected from any one of absent, substituted or unsubstituted C1-C8 alkylene, substituted or unsubstituted C1-C8 heteroalkylene, substituted or unsubstituted C1-C8 cycloalkylene, substituted or unsubstituted C1-C8 heterocycloalkylene, substituted or unsubstituted C6-C8 arylene and substituted or unsubstituted C6-C8 heteroarylene.

In some embodiments of the present application, L is selected from any one of absent, substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 heteroalkylene, substituted or unsubstituted C1-C6 cycloalkylene, substituted or unsubstituted C1-C6 heterocycloalkylene, substituted or unsubstituted C6-C8 arylene and substituted or unsubstituted C6-C8 heteroarylene.

In some embodiments of the present application, L is selected from any one of absent, substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 heteroalkylene, substituted or unsubstituted C1-C6 cycloalkylene, substituted or unsubstituted C1-C6 heterocycloalkylene, substituted or unsubstituted phenylene and substituted or unsubstituted heterophenylene.

In some embodiments of the present application, L is selected from any one of absent, substituted or unsubstituted C1-C4 alkylene, substituted or unsubstituted C1-C4 heteroalkylene, substituted or unsubstituted C1-C4 cycloalkylene, and substituted or unsubstituted C1-C4 heterocycloalkylene.

In some embodiments of the present application, L is selected from any one of absent, substituted or unsubstituted C1-C2 alkylene, substituted or unsubstituted C1-C2 heteroalkylene, substituted or unsubstituted C1-C2 cycloalkylene, and substituted or unsubstituted C1-C2 heterocycloalkylene.

In some embodiments of the present application, R ⁶ is a substituted or unsubstituted trans-cyclooctene group.

In some embodiments of the present application, the orthogonal breaking group is selected from any one of the following structures:

Wherein, L ¹ , L ² , and L ³ are each independently absent, an ester group, an ether group, or a C1-C6 alkylene group;

^R6 is hydrogen or a boronic acid group.

In some embodiments of the present application, L ¹ is selected from any one of absence, ester group, and ether group.

In some embodiments of the present application, ^L2 is absent.

In some embodiments of the present application, L ³ is any one of C1 to C6 alkylene. In some embodiments, L 3 is any one of C1 to C3 alkylene.

In some embodiments of the present application, the orthogonal breaking group is selected from any one of the following:

In some embodiments of the present application, the phosphate group in R ³ can be a monophosphate or a polyphosphate group (eg, 2 or more).

The second aspect of the present application further provides the use of the above nucleotide analogs in the synthesis or sequencing of nucleic acids. In some embodiments of the present application, the synthesis of nucleic acids includes any one of enzymatic synthesis and chemical synthesis.

In some embodiments of the present application, the synthesis of nucleic acid includes any one of enzymatic synthesis and chemical synthesis of DNA.

In some embodiments of the present application, the enzymatic synthesis of nucleic acids refers to the use of the aforementioned compounds as synthesis substrates, a system including a DNA template and an enzyme to complete the synthesis within a set reaction program including temperature conditions and reaction time. In some embodiments, the enzyme in the system includes a DNA polymerase, and in some embodiments, it can be an enzyme that can catalyze a template-dependent DNA polymerization reaction, such as Taq DNA polymerase, Pfu DNA polymerase, etc.

In some embodiments of the present application, chemical synthesis of nucleic acid refers to using the aforementioned compounds as synthetic raw materials, and completing the synthesis process by protecting the non-reactive groups before the reaction, activating the reactive groups, coupling, and deprotecting after the reaction. In some embodiments, chemical synthesis of nucleic acid includes at least one of the phosphodiester bond method, the phosphotriester bond method, and the phosphite amide method. In some embodiments, chemical synthesis of nucleic acid is solid phase synthesis, that is, the synthesis reaction is carried out on a solid phase carrier.

In some embodiments of the present application, the nucleic acid comprises more than 2 nucleotides, further more than 10, 20, 30, 50, 100, 200, 500, 1000 nucleotides.

The third aspect of the present application provides a sequencing method, comprising the following steps:

S1: contacting a template, a polymerase, any of the aforementioned nucleotide analogs and a primer, so that the nucleotide analog is incorporated into the primer in a set order for extension; the nucleotide analog includes four different tags according to different base types;

S2: identifying the incorporated nucleotide analogs based on the type of label on the nucleotide analogs incorporated into the primers;

S3: cleavage of the nucleotide analog using a cleavage agent to expose the 3′-OH;

Repeat steps S1 to S3 to determine the sequence of the template.

In some embodiments of the present application, the cleavage agent includes a tetrazine compound.

In some embodiments of the present application, the tetrazine compound has a general formula as shown in formula (II):

Wherein, R ⁷ and R ⁸ are independently selected from hydrogen, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 heteroalkyl, substituted or unsubstituted C1-C10 cycloalkyl, substituted or unsubstituted C1-C10 heterocycloalkyl, substituted or unsubstituted C6-C10 aryl and substituted or unsubstituted C1-C10 heteroaryl.

In some embodiments of the present application, R ⁷ and R ⁸ are not hydrogen at the same time.

In some embodiments of the present application, the substituents of the C1-C10 alkyl, C1-C10 heteroalkyl, C1-C10 cycloalkyl, C1-C10 heterocycloalkyl, C6-C10 aryl and C1-C10 heteroaryl are selected from at least one of amino and carboxyl groups.

In some embodiments of the present application, ^R7 is hydrogen, and ^R8 is selected from any one of C1~C10 alkyl substituted by amino and/or carboxyl, C1~C10 heteroalkyl substituted by amino and/or carboxyl, C1~C10 cycloalkyl substituted by amino and/or carboxyl, C1~C10 heterocycloalkyl substituted by amino and/or carboxyl, C6~C10 aryl substituted by amino and/or carboxyl, and C1~C10 heteroaryl substituted by amino and/or carboxyl.

In some embodiments of the present application, the detectable label is a fluorescent group.

In some embodiments of the present application, specific examples of fluorescent groups include but are not limited to FAM, HEX, TAMRA, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Texas, ROX, JOE, R6G, EDANS, IFluor, Alexa Fluor, FITC, etc.

In some embodiments of the present application, the nucleotide analogs used for sequencing generally have four different bases. To determine the specific base type, the nucleotide analogs of different base types have different detectable labels. For example, when the detectable label is a fluorescent group, the emission wavelengths of the fluorescent groups of the nucleotide analogs of different base types are significantly different. It is understood that the detectable label can also be other labels known in the art that can distinguish different base types by at least one method in physics, chemistry, and biology.

In the above sequencing process, the cleavage reaction uses pyridazine elimination triggered by IEDDA reaction, and tetrazine reacts with a dienophilic group (such as trans-cyclooctenyl) to form a dihydropyridazine intermediate, which can spontaneously lead to the release of the group at the allylic position through 1,4-elimination. Compared with the existing azide protecting groups, dienophilic groups, especially trans-cyclooctenyl, are more suitable for the production of pyridazine. The group has better stability. At the same time, during the Diels-Alder reaction between tetrazines and diene-philic groups, especially trans-cyclooctene, nitrogen can be released to reduce the tension of the trans-cyclooctene ring, thereby further shortening the excision time and improving the excision efficiency, thereby reducing the time required for sequencing. Taking C ₂ TCO as an example, when using optimized tetrazines containing amino or carboxyl groups as guiding groups for excision, the half-life is only 1.9 seconds, and 99.7% of the excision efficiency can be achieved within 40 seconds.

In a fourth aspect of the present application, a method for synthesizing nucleic acid is also provided, which comprises using the aforementioned nucleotide analogs as synthetic raw materials.

In some embodiments of the present application, the synthesis method is a chemical synthesis method of nucleic acid or an enzymatic synthesis method of nucleic acid.

The fourth aspect of the present application also provides a composition comprising the aforementioned nucleotide analogs.

In some embodiments of the present application, the composition includes sequencing reagents.

In some embodiments of the present application, the composition includes a polymerization reagent.

In some embodiments of the present application, the composition includes a polymerization reaction reagent and a cleavage reaction reagent. The polymerization reaction reagent includes the aforementioned compound, and the cleavage reaction reagent includes a cleavage reagent.

In some embodiments of the present application, the composition includes a polymerization reaction reagent and a cleavage reaction reagent. The polymerization reaction reagent includes an orthogonal cleavage group selected from Any one of the aforementioned compounds, wherein the cleavage reaction reagent includes a tetrazine compound.

In some embodiments of the present application, the polymerization reaction reagent further includes a polymerase.

Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application.

DETAILED DESCRIPTION

The following will clearly and completely describe the concept of the present application and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of them. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present application.

The embodiments of the present application are described in detail below. The described embodiments are exemplary and are only used to explain the present application, and should not be understood as limiting the present application.

In the description of this application, "several" means more than one, "more" means more than two, "greater than", "less than", "exceed" etc. are understood as not including the number itself, "above", "below", "within" etc. are understood as including the number itself, and "about" means within ±20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, etc. If there is a description of first or second, it is only for the purpose of distinguishing the technical features, and cannot be understood as indicating or implying the relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

In the description of the present application, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

Example 1

This example provides a nucleotide analog (Compound 5), the synthesis route of which is as follows:

The specific steps are:

(1) A nucleoside (Compound 1) is reacted with tert-butyldimethylsilyl chloride (TBSCl) and imidazole in N,N-dimethylformamide (DMF) at room temperature for 24 hours to add a TBS protecting group to the 5′-OH to obtain Compound 2.

(2) Under nitrogen protection at room temperature, triphenylphosphine (PPh ₃ ) and trans-cyclooctene-2′-OH were weighed into a round-bottom flask, tetrahydrofuran (THF) was added as a solvent and stirred to dissolve, diethyl azodicarboxylate (DEAD) was slowly added dropwise over 5 min, and then compound 2 dissolved in tetrahydrofuran was slowly added to the above mixture, and the reaction was monitored by TLC. After compound 2 was completely reacted, the reaction solvent was removed by a rotary evaporator, and the intermediate compound 3 was purified by normal silica gel chromatography to obtain a white solid.

(3) Under nitrogen protection at room temperature, compound 3 was weighed into a round-bottom flask, anhydrous THF was added to dissolve it, and then triethylamine hydrogen fluoride (Et ₃ N-HF) was slowly added dropwise. The reaction was stirred and monitored by TLC until all the raw materials were completely reacted. The reaction solvent was removed by rotary evaporator, and it was dissolved in ethyl acetate and extracted and separated with saturated sodium bicarbonate aqueous solution. After the ethyl acetate layer was extracted, saturated sodium chloride was added for extraction, and then dried over anhydrous sodium sulfate, filtered and the ethyl acetate was removed by a rotary evaporator. The obtained yellow oily liquid was purified by normal silica gel chromatography to obtain the intermediate compound 4 as a white solid.

(4) Compound 4 and proton sponge were weighed in a dried two-necked bottle, the air was evacuated and replaced with _N2 for three times, 20 ml of trimethyl phosphate was added and ultrasonically dissolved, and then placed in an ice-water bath, _POCl3 was slowly added dropwise in an ice-water bath, and stirred for reaction for 2 hours in an ice-water bath; another dried two-necked bottle was taken, ammonium pyrophosphate was weighed, the air was evacuated and replaced with _N2 for three times, 20 ml of DMF was added and ultrasonically dissolved, DIPEA was added and placed in an ice-water bath, and then the reaction solution of the first step monophosphoric acid was added back to the above solution, and the ice-water bath was kept for reaction for 2 hours; after 2 hours, 10 ml of 1M TEAB was added dropwise in an ice-water bath to quench, and after reaction for 1 hour, the sample was sent for HPLC analysis. The target molecule compound 5 was purified by DEAE ion exchange chromatography and preparative HPLC, and was confirmed by ¹ H NMR.

Example 2

This example provides a nucleotide analog (Compound 9), the synthesis route of which is as follows:

The specific steps are:

(1) Under nitrogen protection at room temperature, compound 2 was weighed into a round-bottom flask, anhydrous DMF was added as solvent, followed by anhydrous dichloromethane (DCM) and 4-dimethylaminopyridine (DMAP), stirred at room temperature until a uniform liquid was formed, and then carbonyl diimidazole was quickly added. The reaction mixture was resealed and stirred at room temperature for 2 hours until all the raw materials were consumed. The reaction mixture was filtered through silica gel, and the filter cake was washed with ethyl acetate. The solvent was removed by rotary evaporator to obtain a crude compound 6, which was used for the next step reaction.

(2) Under nitrogen protection at room temperature, the crude product of compound 6 was weighed into a round-bottom flask, and trans-cyclooctene-2′-OH and triethylamine were added and stirred for 2 hours. After compound 6 was completely reacted, the reaction solvent was removed by a rotary evaporator and purified by normal silica gel chromatography to obtain the intermediate compound 7 as a white solid.

(3) Under nitrogen protection at room temperature, compound 7 was weighed into a 100 mL round-bottom flask, anhydrous THF was added to dissolve it, and then triethylamine hydrogen fluoride was slowly added dropwise. The reaction was stirred and the reaction was observed by TLC tracking until all the raw materials were completely reacted. The reaction solvent was removed by a rotary evaporator, and it was dissolved in ethyl acetate and extracted and separated with a saturated aqueous sodium bicarbonate solution. After the ethyl acetate layer was extracted, saturated sodium chloride was added for extraction, and then it was dried with anhydrous sodium sulfate. The mixture was filtered and the ethyl acetate was removed by a rotary evaporator. The obtained yellow oily liquid was purified by normal silica gel column chromatography to obtain the intermediate compound 8 as a white solid.

(4) Weigh the intermediate compound 8 and the proton sponge in a 100 mL dried two-necked bottle, evacuate the air, replace it with N ₂ three times, add 20 mL of trimethyl phosphate, ultrasonically dissolve it, put it in an ice-water bath, slowly drop POCl ₃ in the ice-water bath, and stir and react for 2 hours in the ice-water bath; take another dried two-necked bottle, weigh ammonium pyrophosphate, evacuate the air, replace it with N ₂ three times, add 20 mL of DMF, ultrasonically dissolve it, add DIPEA, and put it in an ice-water bath, then add the reaction solution of the first step monophosphoric acid back to the above solution, keep the ice-water bath to react for 2 hours; after 2 hours, drop 10 mL of 1M TEAB in the ice-water bath to quench, react for 1 hour, and send the sample for HPLC analysis. After purification by DEAE ion exchange chromatography and preparative HPLC, the target molecule compound 9 was obtained, which was confirmed by ¹ H NMR.

Example 3

This embodiment provides a tetrazine compound 11 for excision reaction, and its synthesis route is as follows:

The specific preparation process is as follows:

n-boc-b-cyano-l-alanine (compound 10) (600 mg, 2.8 mmol), acetic acid formamidinium salt (1.46 g, 14 mmol), Zn(OTf) ₂ (255 mg, 0.7 mmol), dioxane (8.4 mL, 98 mmol) and hydrazine monohydrate (6.8 mL, 140 mmol) were added to a round-bottom flask and mixed evenly, and the mixture was immediately sealed. Stirred at 30°C for 72 hours, and then poured into 50 mL of ice water. After adding NaNO ₂ (3.9 g, 56 mmol), the solution was acidified with 2N HCI aqueous solution (60 mL). The mixture was extracted with ethyl acetate (5×70 mL), and the organic layer was dried over MgSO ₄ , filtered and concentrated. Purification was performed by preparative reverse phase column chromatography (C18, H ₂ O/MeCN, gradient elution, 0.1% formic acid) to obtain tetrazine (compound 11), which was characterized by ¹ H NMR.

Example 4

This embodiment provides a tetrazine compound 13 for excision reaction, and its synthesis route is as follows:

The specific preparation process is as follows:

3-Cyanpropionic acid (Compound 12, 1 g, 8.26 mmol), acetic acid formamidinium salt (4.3 g, 41.3 mmol), Zn(OTf) ₂ (0.9 g, 2.5 mmol), hydrazine monohydrate (10 mL, 206 mmol) were mixed evenly in a round-bottom flask and sealed immediately. After stirring at 60°C for 60 minutes, NaNO ₂ (2.3 g, 33 mmol) was added, the solution was acidified with 2N HCI aqueous solution, and then the mixture was extracted with ethyl acetate (6×150 mL), and the organic layer was dried over MgSO ₄ , filtered and concentrated. Purification was performed by reverse phase column chromatography (H ₂ O/MeCN, gradient elution, 0.1% formic acid) to obtain tetrazine (Compound 13), which was characterized by ¹ H NMR.

Example 5

This example provides a nucleotide analogue, the synthesis route of which is different from that of Example 1 in that 2→3 is replaced by the following 2→14→15, and then compound 15 is used to replace compound 3 in steps (3) to (4) of Example 1:

The specific preparation process of 2→14→15 is as follows:

(1) Under nitrogen protection at room temperature, compound 2 was dissolved in acetonitrile (MeCN), dibromoethane and K ₂ CO ₃ were added, and the mixture was stirred at 80°C overnight and then cooled to room temperature. Deionized water was added to quench the reaction, and the product was extracted with ether. The organic layer was washed with brine and dried over MgSO ₄ , and the volatiles were removed under reduced pressure. The crude product was purified by column chromatography (30% EtOAc/heptane) to obtain intermediate 14 as a white solid.

(2) Intermediate 14 was dissolved in dry DMSO and purged with _N2 for 10 minutes. Potassium tert-butoxide (t-BuOK) was slowly added in portions and the mixture was stirred for 10 minutes. The mixture was then diluted with EtOAc and quenched with ice water. The layers were separated, the organic layer was washed with deionized water, brine, and dried over _MgSO4 . The volatiles were removed under reduced pressure and the product was dissolved in MeOH. Sodium borohydride ( _NaBH4 ) was added in portions and the mixture was stirred for 1.5 hours. The reaction was quenched with _H2O and the pH was adjusted to 7 with 0.1M HCl. The product was then washed with EtOAc, brine, and dried over _MgSO4 . The volatiles were removed and the crude product was purified by column chromatography (30% EtOAc/heptane) to give compound 15, which was characterized by ^1H NMR.

Example 6

This example provides a nucleotide analogue, the synthesis route of which is different from that of Example 1 in that 2→3 is replaced by 2→16→17→18→19 as follows, and then compound 19 is used to replace compound 3 in steps (3) to (4) of Example 1:

The specific preparation process is as follows:

a) Compound 2 was weighed and dissolved in DMF, and K ₂ CO ₃ was added. Trichloroethylene was added dropwise at 70°C and reacted overnight. After cooling to room temperature, deionized water was added to quench the reaction, and the product was extracted with ether. The combined organic layer was washed with brine and dried with Na ₂ SO ₄ , and the volatiles were removed under reduced pressure. The extraction, washing and drying steps were repeated to obtain Compound 16.

b) Compound 16 was dissolved in ether and n-BuLi was added dropwise. The mixture was stirred at -78°C for 1 hour, then heated to -40°C for 1 hour and stirred again for 1 hour. Deionized water was added to quench the reaction, and the product was extracted with ether. The combined organic layer was washed with saturated NH ₄ Cl and brine, and dried over Na ₂ SO _4. The volatiles were removed under reduced pressure, and the crude product was purified by column chromatography (1% EtOAc/heptane) to give Compound 17.

c) Compound 17 was dissolved in toluene and purged with _N2 for 10 minutes. Pinacolborane and RuHClCO( _PPh3 ) ₃ were added, stirred at 50°C overnight, cooled to room temperature and volatiles were removed under reduced pressure. The crude product was dissolved in ether, washed with saturated _NaHCO3 and _brine , and dried over _Na2SO4 . After removing volatiles under reduced pressure, the crude product was purified by column chromatography (30-40% EtOAc/heptane) to give compound 18, which was characterized by ^1H NMR.

d) Compound 18 was hydrolyzed in PBS to obtain compound 19.

Example 7

This example provides a group of four nucleotide analogs with different detectable labels, including nucleotide analogs dATP, dCTP, dGTP and dTTP. Taking dCTP as an example, the difference between it and the nucleotide analog in Example 1 is that the base is modified with a detectable label through a linking group, specifically an optional fluorescent group Fluor, and the structural formula is as follows:

Similar to dCTP, dATP, dGTP and dTTP are each modified with a fluorescent group via a linker group on the base, and the excitation bands of these fluorescent groups are different.

Example 8

This example provides a group of four nucleotide analogs with different detectable labels, including nucleotide analogs dATP, dCTP, dGTP and dTTP. Taking dCTP as an example, the difference between it and Example 2 is that the base is modified with a detectable label through a linking group, specifically an optional fluorescent group Fluor, and the structural formula is as follows:

Similar to dCTP, dATP, dGTP and dTTP are each modified with a fluorescent group via a linker group on the base, and the excitation bands of these fluorescent groups are different.

Example 9

This example provides a group of four nucleotide analogs with different detectable labels, including nucleotide analogs dATP, dCTP, dGTP and dTTP. Taking dCTP as an example, the difference between it and Example 5 is that the base is modified with a detectable label through a linking group, specifically an optional fluorescent group Fluor, and the structural formula is as follows:

Similar to dCTP, dATP, dGTP and dTTP are each modified with a fluorescent group via a linker group on the base, and the excitation bands of these fluorescent groups are different.

Example 10

This example provides a group of four nucleotide analogs with different detectable labels, including nucleotide analogs dATP, dCTP, dGTP and dTTP. Taking dCTP as an example, the difference between it and Example 6 is that the base is modified with a detectable label through a linking group, specifically an optional fluorescent group Fluor, and the structural formula is as follows:

Similar to dCTP, dATP, dGTP and dTTP are each modified with a fluorescent group via a linker group on the base, and the excitation bands of these fluorescent groups are different.

Example 11: Thermal stability test

The nucleotide analogs prepared in Examples 1, 2, 5, and 6 were respectively prepared into 0.1 mM PBS (pH=9) solutions, heated at 60°C for 5s to 10min, and samples were taken at multiple different time points. The proportion of nucleotides with unblocked 3′-OH was analyzed by HPLC, and the stability of the ^R1 protecting group in Examples 1, 2, 5, and 6 was determined according to the HPLC retention time, peak height, and peak area. According to the results of thermal stability reflected by the degradation rate of the nucleotide analogs at different time points, the ^R1 protecting group of the above nucleotide analogs has good thermal stability, which is 3.4 times higher than the stability of the nucleotides protected by azidomethyl under the same conditions. Therefore, the nucleotide analogs provided in the examples of the present application can ensure the smooth progress of polymerization reaction and signal acquisition in the sequencing cycle, reduce the phasing value, and facilitate the sequencing of the template with high accuracy.

Example 12: Comparison of excision efficiency

THPP was used as a deprotection agent to perform a removal reaction on Comparative Example 1 (azidomethyl-protected nucleotide analogue). Tetrazine compounds 11 and 13 prepared in Examples 5 and 6 were used as deprotection reagents to perform excision reactions on the nucleotide analogs prepared in Examples 1, 2, 5, and 6, and their respective half-retention rates were compared. The principle of the tetrazine excision reaction is shown in the following route:

The results show that the half-retention time of excision of the azidomethyl-protected nucleotide under five times the equivalent of THPP at room temperature is 6.4 min, while the half-retention time of excision using five times the equivalent of tetrazine compound in the example is 4.1 min, and the half-retention rate of excision of the nucleotide analogs of Examples 1, 2, 5, and 6 using compounds 11 and 13 is significantly higher than the half-retention rate of excision of the nucleotide analogs of Comparative Example 1 using THPP. Therefore, using the nucleotide analogs provided in the above examples for primer extension and using the corresponding tetrazine compounds for deprotection can greatly accelerate the excision reaction and shorten the sequencing time.

Example 13: Sequencing Example

This embodiment provides a method for sequencing a human genome, and the specific process is as follows:

1. Sequencing library construction

(1) Nucleic acid extraction: Use a rapid DNA extraction kit (TIANGEN, KG203) to complete the extraction and purification of genomic DNA of the sample. For specific operations, see its operating instructions.

(2) Library construction: Use the VAHTS Universal Plus DNA Library Prep Kit for Illumina (Cat. No. ND617-02) to construct the library. For specific operations, please refer to its operating instructions.

(3) Library quality control: the enriched library is subjected to concentration detection and fragment length quality control.

After the above operation, a library sample of about 50 nM with a length of about 1-1000 bp was obtained.

2. Preparation of sequencing chips

Illumina's MiSeq sequencer and its accompanying sequencing kit (MiSeq Reagent Kit v3) were used for library denaturation, library loading, and chip surface amplification to obtain DNA amplification clusters, and sequencing primers were added. ACACTCTTTCCCTACACGACGCTCTTCCGATC (SEQ ID No. 1). After hybridization is completed, the DNA amplification cluster hybridized with the sequencing primer is fixed in the flow tank of the sequencing chip, waiting for the next sequencing reaction.

3. Sequencing

(1) Preparation of sequencing reagents

A polymerization reaction solution is prepared, which contains DNA polymerase, Mg ²⁺ and 1 μM each of the nucleotide analogs dATP, dCTP, dGTP and dTTP in Example 7; as well as an elution buffer, a pre-wash buffer and an excision reaction solution containing compound 11.

(2) Sequencing reaction cycle

In the amplified chip, the pre-wash buffer and the polymerization reaction solution are pumped in sequence to start the polymerization reaction. When the polymerization reaction solution is finished, the signal of the entire sequencing chip is collected to determine the type of base bound to the primer chain on each amplification cluster. After the signal collection is completed, the elution buffer and the excision reaction solution are pumped in sequence to react, and then the elution buffer is pumped in for cleaning.

Repeat the above steps to perform the next sequencing cycle, for a total of 100 sequencing cycles.

Comparative Example 2: A method for sequencing the human genome is provided, which differs from Example 12 in that the nucleotide analogs in the polymerization reaction solution are replaced by the corresponding nucleotides with azidomethyl protected 3′-OH, and at the same time, THPP is used for compound 11 in the excision reaction solution.

The phasing values, pre-phasing values and Q30 reflecting the sequencing quality of different samples of Example 12 and Comparative Example 2 were compared, and the results are shown in Table 1:

Table 1. Results of testing different samples according to the methods of Example 12 and Comparative Example 2

From the above results, it can be seen that the nucleotide analogs used in the embodiment are more completely removed, and the phasing value and pre-phasing value are significantly lower than the control example. At the same time, the sequencing quality Q30 is also significantly higher than the control example. It can be seen that Example 12 can maintain a relatively high accuracy of sequencing.

Embodiment 14

This embodiment provides a sequencing method, which is different from Embodiment 13 in that the nucleotide analogs used in the polymerization reaction solution are a group of four nucleotide analogs in Embodiment 8.

Embodiment 15

This embodiment provides a sequencing method, which is different from embodiment 13 in that the nucleotides used in the polymerization reaction solution are The analogs are a set of four nucleotide analogs in Example 9.

Example 16

This embodiment provides a sequencing method, which is different from Embodiment 13 in that the nucleotide analogs used in the polymerization reaction solution are a group of four nucleotide analogs in Embodiment 10.

The phasing value and pre-phasing value of the excision reagent in the sequencing of Examples 14 to 16 are similar to those in Example 13, both lower than the corresponding control ratios. Q30 is also similar to that in Example 13, both higher than the control ratios, which will not be repeated here.

From the above examples, it can be seen that the nucleotide analogs provided in the examples of the present application, as a substitute for the reversible terminator, can stably protect the 3′-OH under the sequencing reaction conditions, and are quickly cleaved off to expose the 3′-OH after contact with the cleavage reagent, thereby not only reducing the phasing value in the sequencing process, but also ultimately reducing the error rate, improving the accuracy, and greatly shortening the sequencing time.

Examples 17 to 20

This embodiment provides a method for chemical synthesis of DNA, which is based on the phosphite-amide solid-phase chemical synthesis method on a DNA chip using the nucleotides of Examples 7 to 10 as reaction raw materials or their precursor compounds to synthesize a DNA sequence of a certain length through reaction steps such as deblocking, activation coupling, capping, and oxidation.

The present application is described in detail above in conjunction with the embodiments, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims (13)

The nucleotide analogues have the structural formula (I):
Wherein, R ¹ includes an optional orthogonal breaking group; R ² includes a base; R ³ includes any one of a hydroxyl group and a phosphate group; ^R4 includes any one of hydrogen and ^OR5 , and ^R5 is an optional orthogonal breaking group. The nucleotide analog according to claim 1, characterized in that at least one of R ¹ , R ² , R ³ , and R ⁴ is further connected to a detectable label. The nucleotide analog according to claim 1 or 2, characterized in that the orthogonal cleavage group is a dienophilic group. The nucleotide analog according to claim 3, characterized in that the orthogonal cleavage group is -LR ⁶ ; Wherein, L is selected from any one of non-existent, substituted or unsubstituted C1-C10 alkylene, substituted or unsubstituted C1-C10 heteroalkylene, substituted or unsubstituted C1-C10 cycloalkylene, substituted or unsubstituted C1-C10 heterocycloalkylene, substituted or unsubstituted C6-C10 arylene and substituted or unsubstituted C6-C10 heteroarylene; ^R6 is selected from a substituted or unsubstituted trans-cyclooctene group, a substituted or unsubstituted norbornene group, a substituted or unsubstituted benzonorbornene group, a substituted or unsubstituted vinyl group, a substituted or unsubstituted cyano group, and a substituted or unsubstituted azidophenyl group. The nucleotide analog according to claim 4, characterized in that the orthogonal breaking group is selected from any one of the following:
Wherein, L ¹ , L ² , and L ³ are each independently absent, an ester group, an ether group, or a C1-C6 alkylene group; ^R6 is hydrogen or a boronic acid group. The nucleotide analog according to claim 4 or 5, characterized in that the orthogonal breaking group is selected from any one of the following:
Use of the nucleotide analogue according to any one of claims 1 to 6 in the sequencing or synthesis of nucleic acids. The sequencing method comprises the following steps: S1: contacting a template, a polymerase, a nucleotide analogue according to any one of claims 1 to 5, and a primer, so that the nucleotide analogue is incorporated into the primer in a set order for extension; the nucleotide analogue is connected with four different detectable labels according to different base types; S2: identifying the incorporated nucleotide analogue according to the type of the tag on the nucleotide analogue incorporated into the primer; S3: using a cleavage agent to cleave the nucleotide analog to expose 3′-OH; Repeat steps S1 to S3 to determine the sequence of the template. The sequencing method according to claim 8, characterized in that the cleavage reagent comprises a tetrazine compound. The sequencing method according to claim 9, characterized in that The tetrazine compound has the general formula (II): Wherein, R ⁷ and R ⁸ are independently selected from any one of hydrogen, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 heteroalkyl, substituted or unsubstituted C1-C10 cycloalkyl, substituted or unsubstituted C1-C10 heterocycloalkyl, substituted or unsubstituted C6-C10 aryl and substituted or unsubstituted C6-C10 heteroaryl. A method for synthesizing nucleic acid, characterized in that it comprises using the nucleotide analogue described in any one of claims 1 to 6 as a synthetic raw material. The method for synthesizing nucleic acid according to claim 11, characterized in that: The synthesis method is chemical synthesis or enzymatic synthesis. A composition, characterized in that it comprises the nucleotide analogue according to any one of claims 1 to 6.