Connecting unit for chemical modification of nucleic acid end group, preparation method of connecting unit and nucleic acid probe

Abstract

The embodiment of the invention provides a connecting unit for chemical modification of a nucleic acid end group, a preparation method of the connecting unit and a nucleic acid probe. The connecting unit for chemical modification of the nucleic acid end group has a triethylene glycol structure, and the connecting unit has the following structure. Therefore, the connecting unit with the triethylene glycol structure is relatively simple in synthesis process, relatively high in yield, efficient in synthesis, good in nucleic acid end group chemical modification effect, applicable to connection of various types of probe molecules and nucleic acid molecules, capable of simply and efficiently preparing nucleic acid probes and wide in application range.

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular to a linking unit for chemical modification of nucleic acid end groups, a preparation method thereof, and a nucleic acid probe. Background Art

[0002] Nucleic acid detection probes have important applications in a variety of industries, including clinical testing, disease prevention and control, environmental monitoring, food safety, and modern agriculture. They are efficient and rapid detection tools. Linkers, which connect nucleic acid sequences to probes, are crucial for improving the efficiency of nucleic acid probe synthesis. Linkers containing triethylene glycol (TEG) units are currently a well-established class of linkers in nucleic acid probes. While the synthesis of these linkers has been reported extensively, they often suffer from complex synthesis processes and low overall yields.

[0003] Therefore, the current linking units used for chemical modification of nucleic acid end groups, their preparation methods, and nucleic acid probes still need to be improved. Summary of the Invention

[0004] The embodiments of the present disclosure provide a linking unit for chemical modification of nucleic acid end groups, a preparation method thereof, and a nucleic acid probe to solve or alleviate one or more technical problems in related technologies.

[0005] As a first aspect of the embodiments of the present disclosure, the embodiments of the present disclosure provide a linking unit for chemical modification of nucleic acid end groups, wherein the linking unit has a triethylene glycol structure and has the following structure: .

[0006] In one possible embodiment, the surface modification groups of the linking unit are carbamate groups, primary hydroxyl groups and secondary hydroxyl groups.

[0007] In one possible embodiment, at least one of the carbamate group and the secondary hydroxyl group of the linking unit can be connected to a probe molecule, and the primary hydroxyl group of the linking unit can be connected to the 3' end or the 5' end of a nucleic acid molecule.

[0008] As a second aspect of the embodiments of the present disclosure, the embodiments of the present disclosure provide a method for preparing the aforementioned linking unit, wherein the method uses triethylene glycol as a raw material and synthesizes the linking unit through the following chemical reaction steps: .

[0009] As a third aspect of the embodiments of the present disclosure, the embodiments of the present disclosure provide a nucleic acid probe, wherein the nucleic acid probe is formed by connecting a probe molecule and a nucleic acid molecule via the aforementioned connecting unit, and the nucleic acid probe has the following structure: ,or , Wherein, R represents the probe molecule, 3' represents the 3' end of the nucleic acid molecule, and 5' represents the 5' end of the nucleic acid molecule.

[0010] In one possible embodiment, the probe molecule is cholesterol, and the nucleic acid probe has the following structure: ,or .

[0011] In one possible embodiment, the types of probe molecules include at least one of the following: rhodamine fluorescent dyes, cyanine fluorescent dyes, coumarin fluorescent dyes, pyrene fluorescent dyes, monoazobenzene quenching units, disazobenzene quenching units, biotin compounds, cholesterol and steroid compounds, chain terpenoid compounds, cyclic terpenoid compounds, ferrocene and modified ferrocene compounds.

[0012] In one possible embodiment, the nucleic acid molecule is a polynucleotide formed by polymerization of one or more nucleotides, and the nucleotides include at least one of the following: Natural and non-natural nucleic acids modified on the sugar ring, wherein the structure of the sugar ring includes at least one of the following: natural deoxyribose, natural ribose, 2'-methoxy substituted deoxyribose, 2'-fluoro substituted deoxyribose, 2'-methoxyethyl substituted deoxyribose, 2'-methylamino-2-oxoethyl substituted deoxyribose, 2'-propargyloxy substituted deoxyribose, 2'-butoxy substituted deoxyribose, 2'-hexyloxy substituted deoxyribose, 2'-octyloxy substituted deoxyribose, 2'-decyloxy substituted deoxyribose, 2'-dodecyloxy substituted deoxyribose, 2'-tetradecyloxy substituted deoxyribose, 2'- Hexadecyloxy-substituted deoxyribose, 2'-octadecyloxy-substituted deoxyribose, 2'-eicosyloxy-substituted deoxyribose, 2'-oxy-4' carbonyl methylene lock-configuration deoxyribose, 2'-oxy-4' carbonyl vinyl lock-configuration deoxyribose, 2'-oxy-4' carbonyl-S-configuration ethyl lock-configuration deoxyribose, 2'-oxy-4' carbonyl-R-configuration ethyl lock-configuration deoxyribose, 2'-fluoro-substituted arabinose, 2'-methoxy-substituted arabinose, L-configuration deoxyribose, L-configuration ribose, deoxyribose with flipped 3' and 5' links, and ribose with flipped 3' and 5' links; Natural and non-natural nucleic acids modified on bases, wherein the bases include at least one of the following: uracil, thymine, cytosine, adenine, guanine, hypoxanthine, 5-methylcytosine, 5-hydroxymethylcytosine, 6-methyladenine, 2,6-diaminopurine, 2-thiouracil, 4-thiouracil, dihydrouracil, tetrahydrouracil, pseudouridine, N-methylpseudouridine; Natural and non-natural nucleic acids modified on nucleotide linking units, wherein the nucleotide linking units include at least one of the following: a racemic phosphorothioate diester bond, an R-configured phosphorothioate diester bond, an S-configured phosphorothioate diester bond, monomethyl hypophosphite, monoethyl hypophosphite, monopropyl hypophosphite, monobutyl hypophosphite, monopentyl hypophosphite, monohexyl hypophosphite, monoheptyl hypophosphite, and monooctyl hypophosphite; Non-natural structure modified nucleic acid linking nucleosides and phosphates: 2',3'-dideoxy-β-D-pyranose glucopyranosyl, wherein the base is at the 6-β position; 2-hydroxymethylmorpholine, wherein the base is at the 6-position; 1-hydroxy-S-2-hydroxy-3-methylene unit, wherein the base is at the 3-position; 1-hydroxy-R-2-hydroxy-3-methylene unit, wherein the base is at the 3-position; S-1,3-dihydroxymethyl-2-methylene unit, wherein the base is at the 2-position; R-1,3-dihydroxymethyl-2-methylene unit, wherein the base is at the 2-position; and an unlocked nucleic acid unit, wherein the structure of the unlocked nucleic acid unit is an RNA-like structure without a chemical bond between the 2' and 3' positions, wherein the base is at the 1'-β position.

[0013] In one possible embodiment, the polynucleotide has a length of 2-240 nucleotides.

[0014] In one possible embodiment, the polynucleotide has a length of 15-100 nucleotides. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the multiple drawings represent the same or similar components or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings only depict some embodiments according to the present disclosure and should not be regarded as limiting the scope of the present disclosure.

[0016] Figure 1 Schematic diagram of a method for preparing a connecting unit according to one embodiment of the present disclosure; Figure 2 is a mass spectrum of compound 3 in the method for preparing a linker unit according to an embodiment of the present disclosure; Figure 3 is a mass spectrum of compound 7 in the method for preparing a linker unit according to an embodiment of the present disclosure; Figure 4Schematic diagram of a method for preparing a cholesterol phosphoramidite monomer (Compound 10) containing a linker unit according to an embodiment of the present disclosure; Figure 5 is a hydrogen nuclear magnetic resonance spectrum of a cholesterol phosphoramidite monomer (Compound 10) containing a linker unit according to an embodiment of the present disclosure; Figure 6 is a mass spectrum of a cholesterol phosphoramidite monomer (Compound 10) containing a linker unit according to an embodiment of the present disclosure; Figure 7 is a mass spectrum of the T20 sequence modified with a cholesterol-TEG linker unit according to an embodiment of the present disclosure; Figure 8 4 is a high performance liquid chromatography spectrum of the T20 sequence modified with a cholesterol-TEG linker unit according to an embodiment of the present disclosure. DETAILED DESCRIPTION

[0017] In the following, only certain exemplary embodiments are briefly described. As will be appreciated by those skilled in the art, the described embodiments may be modified in various ways without departing from the spirit or scope of the present disclosure, and different embodiments may be combined in any manner without conflict. Therefore, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0018] As a first aspect of the embodiments of the present disclosure, the embodiments of the present disclosure provide a linking unit for chemical modification of nucleic acid end groups, wherein the linking unit has a triethylene glycol structure. Specifically, the linking unit has the following structure: .

[0019] Therefore, the synthesis process of the linker unit with a triethylene glycol structure is relatively simple and has a high yield, the synthesis is efficient, the effect of chemical modification of nucleic acid end groups is good, and it can be applied to the connection of various types of probe molecules and nucleic acid molecules, thereby simply and efficiently preparing nucleic acid probes, and has a wide range of applications. For example, the linker unit is suitable for chemical synthesis of natural and non-natural nucleic acids with various luminescent, quenching and functionalized 3'-end modifications, including DNA, RNA, artificially synthesized nucleotides containing non-natural nucleotide units, artificially synthesized nucleotides containing natural and non-natural nucleotide units, etc.

[0020] The inventors have discovered that linkers containing triethylene glycol (TEG) units are currently a highly effective type of linker in nucleic acid probes. While the synthesis of this type of linker has been reported extensively, existing TEG-based linkers suffer from complex synthesis processes and low overall yields. In this application, through in-depth research and development, the inventors have proposed a novel linker with a TEG structure and a method for its preparation. This TEG-based linker exhibits a relatively simple synthesis process with high yield and high synthesis efficiency, is highly effective for chemical modification of nucleic acid end groups, and is suitable for linking a variety of probe molecules to nucleic acid molecules, enabling the simple and efficient preparation of nucleic acid probes with a wide range of applications.

[0021] In one possible embodiment, the surface modification groups of the linking unit can be carbamate groups, primary hydroxyl groups, and secondary hydroxyl groups. Thus, the linking unit containing a triethylene glycol structure designed in this application has multiple different types of surface modification groups, which can be easily connected to nucleic acid molecules and can easily chemically modify the nucleic acid end groups. Specifically, at least one of the carbamate groups and secondary hydroxyl groups of the linking unit can be connected to the probe molecule, and the primary hydroxyl group of the linking unit can be connected to the 3' end or the 5' end of the nucleic acid molecule. In turn, the nucleic acid molecule and the probe molecule can be easily connected together to form a nucleic acid probe with multiple functions.

[0022] As a second aspect of the embodiment of the present disclosure, the embodiment of the present disclosure provides a method for preparing the aforementioned connecting unit, specifically, referring to Figure 1 This method uses triethylene glycol (compound 1) as a raw material and synthesizes a linker unit (compound 9) through the following chemical reaction steps: , The synthesis conditions for each synthesis step can be: (a) sodium hydride, benzyl bromide, tetrahydrofuran; (b) aqueous sodium hydroxide solution, acrylonitrile; (c) borane dimethyl sulfide; (d) 4,4-dimethoxytriphenylmethane chloride, pyridine; (e) 5% palladium-carbon catalyst, methanol, hydrogen; (f) glycerol acetone acetal p-toluenesulfonate; (g) 80% aqueous acetic acid, reflux; fluorenylmethyloxycarbonyl succinimide, saturated aqueous sodium bicarbonate solution, acetonitrile; 80% aqueous acetic acid, reflux; (h) 4,4-dimethoxytriphenylmethane chloride, pyridine. This preparation process is relatively simple and has a high yield.

[0023] In some embodiments of the present application, a specific method for synthesizing a linker unit (Compound 9) by multi-step chemical reaction using triethylene glycol (Compound 1) as a raw material includes: (a) Synthesis of compound 2 Specifically, sodium hydride (24.7 g) was dispersed in 100 ml of tetrahydrofuran, and triethylene glycol (123 g) was dissolved in 250 ml of tetrahydrofuran. The mixture was then added dropwise to the sodium hydride solution with stirring. After heating to reflux, a solution of benzyl bromide (35.0 g) in 100 ml of tetrahydrofuran was then added dropwise. The mixture was heated to reflux at 90°C for 2 hours, monitored by a sampling plate, and developed with a 10:1 volume ratio of dichloromethane to tetrahydrofuran. The reaction was complete. The sodium hydride was then quenched with 10 ml of water. The solvent was then dried, and 6 mol / L hydrochloric acid was added to a pH of 1. The organic phase was extracted with 300 ml of dichloromethane. The organic phase was washed once with water and dried over anhydrous sodium sulfate. The desiccant was filtered out, and the solvent was concentrated to obtain a light yellow oily product, which was used directly in the next step without purification.

[0024] (b) Synthesis of compound 3 Specifically, compound 2 (10 g) was dissolved in a 45% aqueous solution of sodium hydroxide (2.2 g), stirred at 0°C, 0.1 g of hydroquinone was added, acrylonitrile (4.5 g) was added, and the mixture was reacted at room temperature for 1 hour. After the reaction, 30 ml of saturated aqueous ammonium chloride solution and 200 ml of ethyl acetate were added for extraction. The organic phase was washed with 50 ml of saturated aqueous ammonium chloride solution and dried over anhydrous sodium sulfate to obtain 10 g of an oily substance. Column chromatography was performed on 200-300 mesh silica gel and the eluent was n-heptane-n-heptane:ethyl acetate [volume ratio 10:1-3:1] to obtain 7.2 g of a light yellow oily product with a yield of 60%. Specifically, the mass spectrum of compound 3 synthesized in this step is shown in FIG. Figure 2 The molecular formula of compound 3 is C 16 H 23 NO4, theoretical molecular weight 293.1727, measured molecular weight 294.1699 (M + H) + , 311.1965 (M + NH4) + .

[0025] (c) Synthesis of compound 4 Specifically, compound 3 (5.6 g) was dissolved in 35 ml of tetrahydrofuran, and a 10 mol / L solution of borane-dimethyl sulfide in tetrahydrofuran (2 ml) was added. The reaction was determined to be complete after 2 hours. The reaction was quenched by the addition of 2 mol / L hydrochloric acid, and the pH was adjusted to 7 with 1 mol / L sodium hydroxide solution. The solvent was concentrated to yield 5.0 g of the crude product. Purification by column chromatography using 200-300 mesh silica gel and a 5:1 volume ratio of dichloromethane to methanol as the eluent yielded 4.4 g of the product as a pale yellow oil, a yield of 78%.

[0026] (d) Synthesis of compound 5 Specifically, compound 4 (4.5 g) was dissolved in 40 ml of anhydrous pyridine, and 4,4-dimethoxytriphenylmethane (5.3 g) was added. The mixture was allowed to react overnight at room temperature. After completion, 1 ml of methanol was added to quench the reaction. The solvent was then dried, followed by washing with 100 ml of ethyl acetate and 100 ml of saturated sodium bicarbonate solution. The organic phase was then dried over anhydrous sodium sulfate. After filtering out the desiccant, the product was concentrated to a viscous yellow oily liquid, which was used directly in the next step without purification.

[0027] (e) Synthesis of compound 6 Specifically, compound 5 (9 g) was dissolved in 10 ml of methanol, and 5% palladium on carbon (0.5 g) was added. The hydrogen atmosphere was replaced three times, maintaining the hydrogen pressure at 1.2-1.3 atmospheres. The reaction was allowed to proceed overnight at room temperature. The next day, plate chromatography confirmed complete conversion. After filtering out the catalyst, the filtrate was concentrated. Purification was performed by column chromatography using 200-300 mesh silica gel and an eluent of n-heptane-n-heptane:dichloromethane (volume ratio 10:1-1:6) to obtain 6.6 g of the product as a pale yellow oil, with a two-step yield of 92%.

[0028] (f) Synthesis of compound 7 Specifically, compound 6 (5 g) was dissolved in 100 ml of anhydrous tetrahydrofuran, sodium hydride (0.4 g) was added, and the mixture was stirred at room temperature for 2 hours. Then, acetone glycerol p-toluenesulfonate (4.1 g) was added, and the temperature was raised to 40°C and stirred overnight under nitrogen protection. The next day, the plate layer detected that the conversion was complete, and saturated ammonium chloride was added to quench the reaction. The solvent was concentrated, and 100 ml of dichloromethane and 100 ml of water were added for extraction. The organic phase was separated and concentrated to obtain a yellow-brown oily liquid, which was directly used in the next step of synthesis without purification. Specifically, the mass spectrum of compound 7 synthesized in this step is shown in FIG. Figure 3 The molecular formula of compound 7 is C 36 H 49 NO8, the theoretical molecular weight is 623.3458, the measured molecular weight is 646.3346 (M + Na) + .

[0029] (g) Synthesis of compound 8 Specifically, the crude product of compound 7 was added to 60 ml of 80% acetic acid aqueous solution and heated to reflux for 4 hours under nitrogen protection. The acetic acid was removed under reduced pressure, and 100 ml of xylene was added to remove water azeotropically to obtain a brown viscous oily liquid. The crude product was dissolved in 120 ml of acetonitrile and 20 ml of saturated sodium bicarbonate aqueous solution, 4.9 g of fluorenylmethyloxycarbonyl succinimide was added, and stirred at room temperature overnight. The organic solvent was evaporated under reduced pressure, 80 ml of acetic acid was added, and the mixture was heated to reflux for 4 hours under nitrogen protection. All solvents were evaporated under reduced pressure to obtain a yellow-brown viscous oily liquid. Column chromatography purification, 200-300 mesh silica gel, eluent dichloromethane-dichloromethane: methanol [volume ratio 20:1-5:1], obtained 2.3 g of light yellow oily product, two-step yield 45%.

[0030] (h) Synthesis of compound 9 Specifically, compound 8 (2 g) was dissolved in 50 ml of anhydrous pyridine and stirred at 0°C for 15 minutes. Under nitrogen, a solution of 1.4 g of 4,4-dimethoxytriphenylmethane in 10 ml of anhydrous pyridine was slowly added dropwise to the solution, which was then allowed to warm to room temperature. The mixture was stirred overnight under nitrogen. The next day, 1 ml of methanol was added to quench the reaction. The solvent was evaporated under reduced pressure, and the crude product was dissolved in 100 ml of dichloromethane, washed with 100 ml of saturated sodium bicarbonate, and dried over anhydrous sodium sulfate. The crude product was purified by column chromatography using 200-300 mesh silica gel and an eluent of dichloromethane:methanol (volume ratio 30:1-7:1) to obtain 1.6 g of the product as a pale yellow oil in a 51% yield. Specifically, the H NMR spectrum data of compound 9 synthesized in this step are: 1H-NMR (CDCl3) δ ppm: 7.73-5.75 (d, ArH, 2H), 7.57-5.59 (d, ArH, 2H),7.35-7.42 (m, ArH, 4H), 7.17-7.30 (m, ArH, 9H), 6.78-6.80 (m, ArH, 4H), 4.35-4.36 (m, CHO, 2H), 4.09-4.19 (m, CHO, 1H), 3.93-3.99 (m, CHO, 1H), 3.75 (s, CHO, 6H), 3.48-3.64 (m, CHO, 14H), 3.26-3.31 (m, CH2O, 2H), 3.11-3.20 (m, CH2O, 2H), 1.68-1.80 (m, CH2, 2H).

[0031] As a third aspect of the embodiments of the present disclosure, the embodiments of the present disclosure provide a nucleic acid probe, which is formed by connecting a probe molecule and a nucleic acid molecule through the aforementioned connecting unit, and the nucleic acid probe has the following structure: ,or , Here, R represents the probe molecule, 3' represents the 3' end of the nucleic acid molecule, and 5' represents the 5' end of the nucleic acid molecule. Thus, the linker unit having an ethylene glycol structure in the embodiment of the present application can be used to easily connect the probe molecule and the nucleic acid molecule to form a nucleic acid probe, which has a wide range of applications.

[0032] According to some embodiments of the present application, the types of probe molecules may include at least one of the following: rhodamine fluorescent dyes, cyanine fluorescent dyes, coumarin fluorescent dyes, pyrene fluorescent dyes, monoazobenzene quenching units, disazobenzene quenching units, biotin compounds, cholesterol and steroid compounds, chain terpenoid compounds, cyclic terpenoid compounds, ferrocene and modified ferrocene compounds.

[0033] According to some embodiments of the present application, the nucleic acid molecule may be a polynucleotide polymerized from one or more nucleotides, and the nucleotides may include at least one of the following: Natural and non-natural nucleic acids modified on the sugar ring, the structure of the sugar ring may include at least one of the following: natural deoxyribose, natural ribose, 2'-methoxy substituted deoxyribose, 2'-fluoro substituted deoxyribose, 2'-methoxyethyl substituted deoxyribose, 2'-methylamino-2-oxyethyl substituted deoxyribose, 2'-propargyloxy substituted deoxyribose, 2'-butoxy substituted deoxyribose, 2'-hexyloxy substituted deoxyribose, 2'-octyloxy substituted deoxyribose, 2'-decyloxy substituted deoxyribose, 2'-dodecyloxy substituted deoxyribose, 2'-tetradecyloxy substituted deoxyribose, 2' - hexadecyloxy-substituted deoxyribose, 2'-octadecyloxy-substituted deoxyribose, 2'-eicosyloxy-substituted deoxyribose, 2'-oxy-4' carbonyl methylene lock-configuration deoxyribose, 2'-oxy-4' carbonyl vinyl lock-configuration deoxyribose, 2'-oxy-4' carbonyl-S-configuration ethyl lock-configuration deoxyribose, 2'-oxy-4' carbonyl-R-configuration ethyl lock-configuration deoxyribose, 2'-fluoro-substituted arabinose, 2'-methoxy-substituted arabinose, L-configuration deoxyribose, L-configuration ribose, deoxyribose with flipped 3' and 5' links, ribose with flipped 3' and 5' links; Natural and non-natural nucleic acids modified on bases, the bases may include at least one of the following: uracil, thymine, cytosine, adenine, guanine, hypoxanthine, 5-methylcytosine, 5-hydroxymethylcytosine, 6-methyladenine, 2,6-diaminopurine, 2-thiouracil, 4-thiouracil, dihydrouracil, tetrahydrouracil, pseudouridine, N-methylpseudouridine; Natural and non-natural nucleic acids modified on nucleotide linking units, wherein the nucleotide linking units include at least one of the following: a racemic phosphorothioate diester bond, an R-configured phosphorothioate diester bond, an S-configured phosphorothioate diester bond, monomethyl hypophosphite, monoethyl hypophosphite, monopropyl hypophosphite, monobutyl hypophosphite, monopentyl hypophosphite, monohexyl hypophosphite, monoheptyl hypophosphite, and monooctyl hypophosphite; Non-natural structure modified nucleic acid linking nucleosides and phosphates: 2',3'-dideoxy-β-D-pyranose glucopyranosyl, wherein the base is at the 6-β position; 2-hydroxymethylmorpholine, wherein the base is at the 6-position; 1-hydroxy-S-2-hydroxy-3-methylene unit, wherein the base is at the 3-position; 1-hydroxy-R-2-hydroxy-3-methylene unit, wherein the base is at the 3-position; S-1,3-dihydroxymethyl-2-methylene unit, wherein the base is at the 2-position; R-1,3-dihydroxymethyl-2-methylene unit, wherein the base is at the 2-position; and an unlocked nucleic acid unit, wherein the structure of the unlocked nucleic acid unit is an RNA-like structure without a chemical bond between the 2' and 3' positions, wherein the base is at the 1'-β position.

[0034] According to some embodiments of the present application, the length of the polynucleotide may be 2-240 nucleotides; specifically, the length of the polynucleotide may be 15-100 nucleotides.

[0035] According to a specific embodiment of the present application, the probe molecule may be cholesterol, and the nucleic acid probe may have the following structure: ,or .

[0036] Specifically, refer to Figure 4 , the linking unit having a triol structure (compound 9) in the examples of the present application can be first used to prepare a cholesterol phosphoramidite monomer (compound 10, hereinafter abbreviated as "cholesterol-TEG linking unit") containing the linking unit.

[0037] .

[0038] Specifically, the steps of synthesizing a cholesterol phosphoramidite monomer containing the linker unit (Compound 10, i.e., cholesterol-TEG linker unit) may include: Compound 9 (1.6 g) was dissolved in 20 ml of dichloromethane, and 1 ml of piperidine was added. The mixture was stirred at room temperature under nitrogen for 1 hour. After the reaction, the solvent was removed by concentration. The crude product was washed with n-hexane (5 ml each) for a total of five washes. The resulting pale yellow oily liquid was dissolved in 20 ml of anhydrous dichloromethane, and 2 ml of triethylamine and cholesteryl chloroformate (1.1 g) were added. After stirring at room temperature for 2 hours, 0.1 ml of methanol was added. The solvent was removed by concentration under reduced pressure. The crude product was dissolved in 20 ml of ethyl acetate, washed with 20 ml of saturated sodium bicarbonate solution, and the organic phase was dried over anhydrous sodium sulfate. The desiccant was filtered off and concentrated to obtain a pale yellow waxy solid. After dissolving in 2 ml of dichloromethane, the solid was added dropwise to 25 ml of methyl tert-butyl ether at 0°C, resulting in a pale yellow precipitate. Filtering gave 2.0 g of a pale yellow solid.

[0039] The solid was dissolved in 10 ml of anhydrous dichloromethane, and 4,5-dicyanoimidazole (0.5 g) was added. After nitrogen displacement, N,N-diisopropyl-O-cyanoethylphosphoramidite (0.5 g) was added and stirred at room temperature for 2 hours. A 1 mol / L aqueous sodium carbonate solution was added for washing, and the organic phase was dried over anhydrous sodium sulfate. After filtering out the desiccant, the mixture was concentrated to obtain a light yellow waxy solid in an 82% yield. Specifically, the H NMR spectrum of compound 10 synthesized in this step is shown in FIG. Figure 5 , mass spectrum reference Figure 6 The molecular formula of compound 10 is C 70 H 106 N3O 11 P, theoretical molecular weight is 1195.7565, measured molecular weight is 897.4131 (M-DMT+Na) + .

[0040] According to some embodiments of the present application, as described above, after preparing the cholesterol-TEG linker unit (Compound 10), Compound 10 can be connected to the 3' end or 5' end of the nucleic acid molecule to obtain the aforementioned cholesterol nucleic acid probe.

[0041] According to some specific embodiments of the present application, the aforementioned cholesterol nucleic acid probe can be prepared by the following method: (1) The experimental conditions for nucleic acid synthesis and modification can be: The test environment temperature is 25±1°C and the humidity is 10%~30%.

[0042] (2) The methods and steps for nucleic acid synthesis and modification may be: Nucleic acid sequence information: 5'-XTTTTTTTTTTTTTTTTTTTT-3' The nucleic acid structure is DNA, and the sequence is T20 composed of 20 thymidine deoxynucleotides, wherein X represents a cholesterol monomer modified with a linking unit.

[0043] Synthesis experiment instruments and reagents: The nucleic acid synthesizer manufacturer is Beijing Haijing Gaochuang Technology Co., Ltd., and the model number is HJ01201202. The nucleic acid synthesis capacity is 200 nanomoles. The synthesis column is a porous glass microsphere powder packing column modified with a universal structure. The concentration of deoxythymidine phosphoramidite monomer is 0.05 mmol / L, and the water content of the elution acetonitrile is 10-15 ppm. The deprotection reagent is a 3% (w / v) solution of trichloroacetic acid in dichloromethane. The activator is a 0.45 mol / L solution of 5-ethylthiotetrazole in acetonitrile. The oxidant is a 0.05 mol / L tetrahydrofuran-water-pyridine mixture (with a volume ratio of tetrahydrofuran:water:pyridine of 90:5:5). Capping Reagent A is a 10% (w / v) solution of acetic anhydride in tetrahydrofuran, and Capping Reagent B is a 5% (w / v) solution of 1-methylimidazole in tetrahydrofuran. Cholesterol monomers modified with linker units were dissolved in anhydrous acetonitrile at a concentration of 0.075 mmol / L.

[0044] Nucleic acid synthesis was performed using the DMT-off method. After completion, the porous glass microspheres were removed and placed in 1 mL of 28% ammonia for aminolysis at 60°C for 2 hours. The aminolysis solution was then drained and analyzed using high-performance liquid chromatography.

[0045] Analytical instruments and reagents: The HPLC model was Waters 2695, the mobile phase A was chromatographically pure acetonitrile, the mobile phase B was 10% triethylamine acetate in deionized water, the analytical column model was C18 Waters X-Bridge, the particle size was 5 um, the size was 250 mm × 4.6 mm, the flow rate was 0.2 mL / min, and the column temperature was 25°C.

[0046] The mass spectrometer was Shimadzu LCMS-IT / TOF.

[0047] Specifically, the mass spectrum of the T20 sequence modified with the cholesterol-TEG linker prepared by the above method is referenced to Figure 7 The theoretical molecular weight of the T20 sequence modified with a cholesterol-TEG linker unit is 6776.97, and the detected molecular weight is 6776.2, which meets the requirements.

[0048] Reference HPLC chromatogram of T20 sequence modified with cholesterol-TEG linker Figure 8 , where the horizontal axis is the retention time (min), the results analysis refers to Table 1:

[0049] From the data in Table 1 , it can be seen that the T20 sequence modified with the cholesterol-TEG linker unit meets the requirements.

[0050] Therefore, the linking unit with a triethanol structure designed and prepared in the present application can better connect the probe molecule and the nucleic acid molecule, has a good nucleic acid end group chemical modification effect, is simple and efficient to synthesize, and has a wide range of applications.

[0051] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of such features. Throughout the present disclosure, "plurality" means two or more, unless otherwise specifically defined.

[0052] In this disclosure, unless otherwise expressly specified or limited, terms such as "mounted," "connected," "connect," and "fixed" should be understood broadly. For example, they may refer to fixed connections, detachable connections, or integration; mechanical connections, electrical connections, or communication; direct connections or indirect connections through an intermediate medium; and internal connections between two components or interactions between two components. Those skilled in the art will understand the specific meanings of these terms in this disclosure based on specific circumstances.

[0053] In the present disclosure, unless otherwise expressly specified and limited, a first feature being “above” or “below” a second feature may include the first and second features being in direct contact, or may include the first and second features being in contact not directly but through another feature between them. Moreover, a first feature being “above”, “above”, and “above” a second feature includes the first feature being directly above and obliquely above the second feature, or simply indicates that the first feature is at a higher level than the second feature. A first feature being “below”, “below”, and “below” a second feature includes the first feature being directly above and obliquely above the second feature, or simply indicates that the first feature is at a lower level than the second feature. The above disclosure provides many different embodiments or examples for realizing different structures of the present disclosure. In order to simplify the present disclosure, the components and settings of specific examples are described above. Of course, they are merely examples and are not intended to limit the present disclosure. In addition, the present disclosure may repeat reference numbers and / or reference letters in different examples. Such repetition is for the purpose of simplicity and clarity and does not in itself indicate the relationship between the various embodiments and / or settings discussed.

[0054] The above are only specific embodiments of the present disclosure, but the scope of protection of the present disclosure is not limited thereto. Any person skilled in the art can easily conceive of various modifications or substitutions within the technical scope disclosed in this disclosure, and such modifications or substitutions should be included in the scope of protection of the present disclosure. Therefore, the scope of protection of the present disclosure should be based on the scope of protection of the claims.

Claims

1. A linking unit for chemical modification of nucleic acid end groups, characterized in that: The linking unit has a triethylene glycol structure, and the linking unit has the following structure: 。 2. The connection unit according to claim 1, wherein: The surface modification groups of the linking unit are carbamate groups, primary hydroxyl groups and secondary hydroxyl groups.

3. The connection unit according to claim 2, characterized in that At least one of the carbamate group and the secondary hydroxyl group of the linking unit can be connected to a probe molecule, and the primary hydroxyl group of the linking unit can be connected to the 3' end or the 5' end of a nucleic acid molecule.

4. A method for preparing the connecting unit according to any one of claims 1 to 3, characterized in that: include: Triethylene glycol is reacted with benzyl bromide in an alkaline tetrahydrofuran solution to obtain compound 2; The compound 2 is dissolved in an alkaline aqueous solution, and hydroquinone and acrylonitrile are added to react to obtain the compound 3; The compound 3 is dissolved in tetrahydrofuran and mixed with a tetrahydrofuran solution of borane-dimethyl sulfide to react to obtain compound 4; The compound 4 was reacted with 4,4-dimethoxytriphenylmethane at room temperature overnight to obtain compound 5; The compound 5 was dissolved in methanol, palladium-carbon catalyst was added, and the mixture was reacted at room temperature overnight in a hydrogen atmosphere to obtain compound 6; Compound 6 and sodium hydride were mixed and stirred, glycerol acetone acetal toluenesulfonate was added, and the mixture was heated under an inert atmosphere to react to obtain compound 7; Compound 7 is added to an aqueous acetic acid solution, heated under reflux under an inert atmosphere to remove the acetic acid to obtain a purified crude product, which is dissolved in acetonitrile and a saturated aqueous sodium bicarbonate solution, and fluorenylmethoxycarbonyl succinimide is added for reaction. After removing the organic solvent, acetic acid is added, and the mixture is heated under reflux under nitrogen protection to obtain compound 8; The compound 8 is dissolved in anhydrous pyridine, 4,4-dimethoxytriphenylmethane is added under the protection of an inert atmosphere, and the reaction is carried out overnight under an inert atmosphere to obtain compound 9, wherein: Compounds 2 to 9 have the following structural formulas: ， ， ， ， ， ， ， 。 5. A nucleic acid probe, characterized in that The nucleic acid probe is formed by connecting a probe molecule and a nucleic acid molecule through the connecting unit according to any one of claims 1 to 3, and the nucleic acid probe has the following structure: ,or ， Wherein, R represents the probe molecule, 3' represents the 3' end of the nucleic acid molecule, and 5' represents the 5' end of the nucleic acid molecule.

6. The nucleic acid probe according to claim 5, wherein The probe molecule is cholesterol, and the nucleic acid probe has the following structure: ,or 。 7. The nucleic acid probe according to claim 5, characterized in that The types of the probe molecules include at least one of the following: rhodamine fluorescent dyes, cyanine fluorescent dyes, coumarin fluorescent dyes, pyrene fluorescent dyes, monoazobenzene quenching units, bisazobenzene quenching units, biotin compounds, cholesterol and steroid compounds, chain terpenoid compounds, cyclic terpenoid compounds, ferrocene and modified ferrocene compounds.

8. The nucleic acid probe according to claim 5, wherein The nucleic acid molecule is a polynucleotide formed by polymerization of one or more nucleotides, wherein the nucleotides include at least one of the following: Natural and non-natural nucleic acids modified on the sugar ring, wherein the structure of the sugar ring includes at least one of the following: natural deoxyribose, natural ribose, 2'-methoxy substituted deoxyribose, 2'-fluoro substituted deoxyribose, 2'-methoxyethyl substituted deoxyribose, 2'-methylamino-2-oxoethyl substituted deoxyribose, 2'-propargyloxy substituted deoxyribose, 2'-butoxy substituted deoxyribose, 2'-hexyloxy substituted deoxyribose, 2'-octyloxy substituted deoxyribose, 2'-decyloxy substituted deoxyribose, 2'-dodecyloxy substituted deoxyribose, 2'-tetradecyloxy substituted deoxyribose, 2'- Hexadecyloxy-substituted deoxyribose, 2'-octadecyloxy-substituted deoxyribose, 2'-eicosyloxy-substituted deoxyribose, 2'-oxy-4' carbonyl methylene lock-configuration deoxyribose, 2'-oxy-4' carbonyl vinyl lock-configuration deoxyribose, 2'-oxy-4' carbonyl-S-configuration ethyl lock-configuration deoxyribose, 2'-oxy-4' carbonyl-R-configuration ethyl lock-configuration deoxyribose, 2'-fluoro-substituted arabinose, 2'-methoxy-substituted arabinose, L-configuration deoxyribose, L-configuration ribose, deoxyribose with flipped 3' and 5' links, and ribose with flipped 3' and 5' links; Natural and non-natural nucleic acids modified on bases, wherein the bases include at least one of the following: uracil, thymine, cytosine, adenine, guanine, hypoxanthine, 5-methylcytosine, 5-hydroxymethylcytosine, 6-methyladenine, 2,6-diaminopurine, 2-thiouracil, 4-thiouracil, dihydrouracil, tetrahydrouracil, pseudouridine, N-methylpseudouridine; Natural and non-natural nucleic acids modified on nucleotide linking units, wherein the nucleotide linking units include at least one of the following: a racemic phosphorothioate diester bond, an R-configured phosphorothioate diester bond, an S-configured phosphorothioate diester bond, monomethyl hypophosphite, monoethyl hypophosphite, monopropyl hypophosphite, monobutyl hypophosphite, monopentyl hypophosphite, monohexyl hypophosphite, monoheptyl hypophosphite, and monooctyl hypophosphite; Non-natural structure modified nucleic acid linking nucleosides and phosphates: 2',3'-dideoxy-β-D-pyranose glucopyranosyl, wherein the base is at the 6-β position; 2-hydroxymethylmorpholine, wherein the base is at the 6-position; 1-hydroxy-S-2-hydroxy-3-methylene unit, wherein the base is at the 3-position; 1-hydroxy-R-2-hydroxy-3-methylene unit, wherein the base is at the 3-position; S-1,3-dihydroxymethyl-2-methylene unit, wherein the base is at the 2-position; R-1,3-dihydroxymethyl-2-methylene unit, wherein the base is at the 2-position; and an unlocked nucleic acid unit, wherein the structure of the unlocked nucleic acid unit is an RNA-like structure without a chemical bond between the 2' and 3' positions, wherein the base is at the 1'-β position.

9. The nucleic acid probe according to claim 8, characterized in that , the length of the polynucleotide is 2-240 nucleotides.

10. The nucleic acid probe according to claim 9, characterized in that , the length of the polynucleotide is 15-100 nucleotides.

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