CN115819474A - A kind of nucleic acid capable of double-terminal conjugated different compounds and its synthesis method and application - Google Patents

Abstract

The invention discloses a nucleic acid capable of double-end conjugation of different compounds, a synthetic method and application thereof. The method comprises the following steps: synthesizing nucleic acid molecules from a 3' end to a 5' end by adopting a solid phase synthesis method to obtain the nucleic acid molecules of which the 3' end is connected to a solid phase carrier, connecting amino to the 5' end of the nucleic acid molecules of which the 3' end is connected to the solid phase carrier, mixing the nucleic acid molecules connected with the amino with activated ester for conjugation reaction, mixing the products of the conjugation reaction with aminolysis solution for aminolysis reaction to obtain the nucleic acid of different compounds capable of being conjugated at two ends, wherein the aminolysis solution contains tert-butylamine and methanol. The invention skillfully designs the preparation process of nucleic acid capable of conjugating different compounds at two ends, overcomes the problems that the amino group at the 3 'end is modified, the amino group at the 5' end needs to be conjugated with activated ester, and the conflict occurs when the 3 'end and the 5' end are connected with specific compounds, increases the sites of a modifying group in a synthetic sequence, provides a brand-new synthetic thought, and promotes the application of molecular biology.

Description

A kind of nucleic acid capable of double-end conjugating different compounds and its synthesis method and application

technical field

The invention belongs to the technical field of molecular biology, and relates to a nucleic acid capable of double-terminally conjugating different compounds and its synthesis method and application.

Background technique

Single-stranded DNA and RNA are composed of a certain number of deoxynucleotides or nucleotides. Some modifications to the structure of DNA or RNA can form nucleic acid tools such as DNA modification probes and RNA modification probes. DNA modification probes and RNA modification probes are widely used in molecular diagnosis QPCR (real-time fluorescence quantitative), STR (short tandem repeats), and FISH (fluorescence in situ hybridization) and other fields.

In general, there are two common approaches that can be used for the engineering of single-stranded DNA and RNA. One is the liquid-phase amino activated ester addition method, and the other is the solid-phase phosphoramidite triester method. The traditional liquid-phase amino activated ester addition method is to dissolve the DNA or RNA containing amino active groups in an alkaline buffer, then add 3 times the molar amount of the modified dye activated ester dissolved in an organic solvent, and react at room temperature for 4 ~12 hours, then add alcohol for precipitation, and wash the precipitate repeatedly with alcohol at the same time to obtain a precipitated solid, which is then dissolved in water, and finally purified by high performance liquid chromatography to obtain a pure DNA modification probe or RNA modification probe. The synthesis process of the solid-phase phosphoramidite triester method mainly includes 4 steps of deprotection, coupling, capping and oxidation. Every time 4 steps are completed, a deoxynucleotide or nucleotide is connected. By repeating these 4 steps DNA or RNA is formed by connecting deoxynucleotides or nucleotides one by one. The solid-phase phosphoramidite triester method can also be used to synthesize modified dyes into DNA or RNA during the synthesis of DNA/RNA. on the RNA strand.

However, in the commonly used transformation methods, there are often conflicts between the 3' end and the 5' end of the nucleic acid when connecting specific compounds. For example, the 5' end amino group needs to be conjugated with an activated ester, while the 3' end still needs to retain the amino activity. When using conventional modification methods, the 5' and 3' amino groups will be modified at the same time, which is difficult to meet the demand.

To sum up, how to provide a method for efficiently synthesizing nucleic acids capable of double-terminally conjugating different compounds is one of the urgent problems in the field of nucleic acid synthesis.

Contents of the invention

Aiming at the deficiencies and actual needs of the prior art, the present invention provides a nucleic acid capable of double-terminal conjugation of different compounds and its synthesis method and application. The present invention designs a synthetic method of nucleic acid capable of double-terminal conjugation of different compounds, which overcomes 3 There is an amino modification at the 'end, the amino group at the 5' end needs to be conjugated with an activated ester, and there will be conflicts between the 3' end and the 5' end when connecting a specific compound.

For reaching above-mentioned purpose, the present invention adopts following technical scheme:

In a first aspect, the present invention provides a method for synthesizing a nucleic acid capable of double-terminally conjugating different compounds, the method comprising:

Synthesize nucleic acid molecules from the 3' end to the 5' end by solid-phase synthesis to obtain a nucleic acid molecule whose 3' end is connected to a solid-phase carrier, and connect an amino group to the nucleic acid molecule whose 3'-end is connected to a solid-phase carrier At the 5' end, the nucleic acid molecule connected to the amino group is mixed with the activated ester to perform a conjugation reaction, and the product of the conjugation reaction is mixed with the ammonium solution to perform an ammonolysis reaction to obtain the nucleic acid that can be conjugated to different compounds at both ends, The ammonia solution contains tert-butylamine and methanol.

The invention ingeniously designs the nucleic acid preparation process that can conjugate different compounds at both ends, realizes that the 3'-terminal amino group can be retained, and the 5'-terminal amino group can be conjugated with the activated ester (NHS), and at the same time, the aminolysis process is further designed to realize Without destroying the amide bond, the nucleic acid molecules can be efficiently separated from the solid phase support and the protective group is removed, which overcomes the modification of the 3' end with the amino group, the 5' end amino group needs to be conjugated with the activated ester, and the 3' end There will be conflicts when connecting a specific compound with the 5' end, increasing the position of the modifying group in the synthetic sequence, providing a new synthesis idea, and the ammonia solution formula is simple and low cost, which promotes the application of molecular biology.

It can be understood that the nucleic acid solid-phase synthesis method commonly used in the art is applicable to the present invention. Specifically, the nucleic acid solid-phase synthesis method can include four steps of deprotection group, coupling, capping and oxidation, and the direction of synthesis is from 3' End to 5' end synthesis, microporous glass beads (Controlledpore glass, CPG) is the synthetic carrier, with a dimethoxytrityl (DMT) protecting group at the end, common A, G, C and T monomers There is a DMT chemical protecting group at the 5' position to protect the 5' hydroxyl (-OH). DMT is unstable under acidic conditions and can be excised with 3% trichloroacetic acid (TCA) to expose the active hydroxyl (-OH). The body is mixed with the activator tetrazole (ACT) to activate the phosphoramidite monomer and undergo a condensation reaction with the hydroxyl group. During the condensation process, the content of water molecules is very high, usually below 30ppm, and the temperature is 25-28 The temperature is better, and dehumidification equipment is required to be installed in the synthetic laboratory. Synthetic column: CPG is used as the solid carrier to which oligonucleotides are attached, and the synthetic column is filled and weighed according to the sample load (μm/g). 7mg of ammonia solution CPG. Cap: In the process of condensation reaction, a small amount of hydroxyl group has not reacted, but it is still active, react with Cap A and Cap B, Cap A: tetrahydrofuran / pyridine / acetic anhydride = 8:1:1 (v/v/ v), Cap B: 16% tetrahydrofuran solution of methylimidazole, the reactant acetamide reacts with the residual hydroxyl group to deactivate the hydroxyl group, terminate the condensation reaction with the subsequent monomer, and avoid base deletion in the synthesized sequence. Oxidation: Use 0.05M iodine solution as an oxidant to oxidize the phosphorus between the bases from 3 to 5 to form a stable phosphodiester bond.

Preferably, the solid phase support includes microporous glass beads (CPG).

It can be understood that all methods for amino modification of nucleic acid terminals commonly used in the art are applicable to the present invention, for example, the synthesis method of phosphoramidite monomers can be used to condense amino groups on the 5' end of the nucleic acid sequence.

Preferably, the activated ester includes succinimide activated ester and/or isothiocyanate activated ester.

Preferably, the succinimide activated ester includes azide-C3-succinimide ester (N3-C3-NHS ester).

Preferably, the ammonia solution also includes water.

Preferably, the volume ratio of tert-butylamine, methanol and water in the ammonia solution is 1:1:(1-10), including but not limited to 1:1:2, 1:1:3, 1:1: 4. 1:1:5, 1:1:7, 1:1:8 or 1:1:9.

Preferably, the conjugation reaction comprises:

The nucleic acid molecules linked to amino groups are mixed with activated ester and alkaline buffer for conjugation reaction.

Preferably, the alkaline buffer comprises sodium bicarbonate solution.

Preferably, the temperature of the conjugation reaction is 30-40°C (for example, 31°C, 32°C, 33°C, 35°C, 36°C, 37°C, 38°C or 39°C), and the time is 3-10h ( For example, it can be 4h, 5h, 6h, 7h, 8h or 9h).

Preferably, the temperature of the ammonolysis reaction is not higher than 60°C.

Preferably, the temperature of the ammonolysis reaction is 50-60°C, including but not limited to 51°C, 52°C, 53°C, 55°C, 56°C, 57°C, 58°C or 59°C.

In the present invention, the temperature of the ammonolysis reaction is controlled, and under the condition of effectively protecting the amide bond in the nucleic acid molecule, an efficient ammonolysis reaction is realized, and the nucleic acid molecule is quickly separated from the solid phase carrier and the protective group on the nucleic acid molecule is removed.

Preferably, a pretreatment step is also included before the ammonolysis reaction.

Preferably, the pretreatment includes:

The product of the conjugation reaction was mixed with the diethylamine-acetonitrile mixture, and the solid was collected.

In the present invention, the pretreatment can effectively remove the β-nitrile ethyl group, prevent the β-nitrile ethyl group from combining with the amino group (-NH2) in the process of ammonolysis, and then improve the yield of the product.

Preferably, the volume ratio of diethylamine to acetonitrile in the diethylamine-acetonitrile mixture is 1:(1-4), including but not limited to 1:2, 1:3 or 1:4.

Preferably, the amino group has a protecting group.

Preferably, the protecting group includes p-methoxytrityl (MMT).

Preferably, the step of removing the protecting group on the amino group is also included before the conjugation reaction.

Preferably, the method for removing the protecting group on the amino group comprises:

The amino acid-linked nucleic acid molecule is mixed with trichloroacetic acid, and the solid is collected.

Preferably, the step of washing with water is also included after the conjugation reaction.

In the present invention, washing with water can effectively remove the residual activated ester, and prevent the residual activated ester from reacting with the 3' terminal amino group during the process of ammonolysis.

As a preferred technical solution, the method for synthesizing a nucleic acid capable of double-terminally conjugating different compounds comprises the following steps:

(1) using solid-phase synthesis to synthesize nucleic acid molecules from the 3' end to the 5' end to obtain a nucleic acid molecule whose 3' end is connected to a solid-phase carrier;

(2) Connect the amino group with a protective group to the 5' end of the nucleic acid molecule whose 3' end is connected to the solid phase carrier, mix the nucleic acid molecule with trichloroacetic acid after connecting the amino group, collect the solid and mix it with the activated ester carry out the conjugation reaction;

(3) Mix the product of the conjugation reaction with the diethylamine-acetonitrile mixture, collect the solid matter and mix it with the ammonium solution to carry out the ammonolysis reaction to obtain the nucleic acid capable of double-terminally conjugating different compounds.

In the second aspect, the present invention provides a nucleic acid capable of double-terminal conjugation to different compounds, the nucleic acid capable of double-terminal conjugation of different compounds is prepared by the method for synthesizing nucleic acids capable of double-terminal conjugation of different compounds described in the first aspect get.

In a third aspect, the present invention provides the use of the nucleic acid capable of double-terminally conjugated to different compounds described in the second aspect in the preparation of nucleic acid probes and/or primers.

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

The invention ingeniously designs the nucleic acid preparation process that can conjugate different compounds at both ends, realizes that the 3'-terminal amino group can be retained, and the 5'-terminal amino group can be conjugated with the activated ester (NHS), and at the same time, the aminolysis process is further designed to realize Without destroying the amide bond, the nucleic acid molecules can be efficiently separated from the solid phase support and the protective group is removed, which overcomes the modification of the 3' end with the amino group, the 5' end amino group needs to be conjugated with the activated ester, and the 3' end There will be conflicts when connecting a specific compound with the 5' end, increasing the position of the modifying group in the synthetic sequence, providing a new synthesis idea, and the ammonia solution formula is simple and low cost, which promotes the application of molecular biology.

Description of drawings

Fig. 1 is the non-ammonolysis reaction MS detection figure of reference substance in embodiment 1;

Fig. 2 is 25 ℃ ammonolysis reaction MS detection figure among the embodiment 1;

Fig. 3 is 30 ℃ ammonolysis reaction MS detection figure in embodiment 1;

Fig. 4 is 37 ℃ ammonolysis reaction MS detection figure in embodiment 1;

Fig. 5 is 50 ℃ ammonolysis reaction MS detection figure in embodiment 1;

Fig. 6 is the MS detection figure of 60 ℃ of ammonolysis reactions in embodiment 1;

Fig. 7 is the MS detection figure of 63 DEG C of ammonolysis reaction in embodiment 1;

Fig. 8 is the MS detection figure of 65 ℃ ammonolysis reaction in embodiment 1;

Fig. 9 is the MS detection chart of 70 ℃ ammonolysis reaction in embodiment 1;

Figure 10 is a structural diagram of N3-C3-NHS ester;

Fig. 11 is the structural diagram of final product in embodiment 2;

Fig. 12 is liquid phase chromatogram detection figure among the embodiment 2;

FIG. 13 is a MS detection diagram in Example 2.

Detailed ways

In order to further illustrate the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with the embodiments and accompanying drawings. It should be understood that the specific implementation manners described here are only used to explain the present invention, rather than to limit the present invention.

If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field, or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that can be purchased through formal channels.

The nucleic acid solid-phase synthesis method in the specific embodiment of the present invention comprises:

The direction of synthesis is from the 3' end to the 5' end. Microporous glass beads (Controlledpore glass, CPG) are the synthetic carrier, with a dimethoxytrityl (DMT) protecting group at the end. Common A, G, C and T monomers have a DMT chemical protection group at the 5' position to protect the 5' hydroxyl (-OH). DMT is unstable under acidic conditions and can be excised by 3% trichloroacetic acid (TCA) to expose the activity. Hydroxyl (-OH), the monomer is mixed with the activator tetrazole (ACT) to activate the phosphoramidite monomer and undergo a condensation reaction with the hydroxyl group. During the condensation process, the content of water molecules is very high, usually at 30ppm Below, the temperature is preferably 25-28 degrees, and dehumidification equipment is required to be installed in the synthetic laboratory;

Synthetic column: CPG is used as the solid carrier to which oligonucleotides are attached, and the synthetic column is filled and weighed according to the sample load (μm/g). Ammonialysis CPG within 7mg;

Cap: In the process of condensation reaction, a small amount of hydroxyl group has not reacted, but it is still active, react with Cap A and Cap B, Cap A: tetrahydrofuran / pyridine / acetic anhydride = 8:1:1 (v/v/ v), Cap B: 16% tetrahydrofuran solution of methylimidazole, the reactant acetamide reacts with the residual hydroxyl group to deactivate the hydroxyl group, terminate the condensation reaction with the subsequent monomer, and avoid base deletion in the synthesized sequence;

Oxidation: Use 0.05M iodine solution as an oxidant to oxidize the phosphorus between the bases from 3 to 5 to form a stable phosphodiester bond.

Amino modifications at the 5' end include:

The amino monomer (MMT-Amino-Modifier C6, CAS: 114616-27-2) is synthesized according to the conventional phosphoramidite monomer method, and the amino group is condensed on the 5' end of the DNA sequence, and the protecting group of MMT remains .

Ammonolysis pretreatment:

Add 20% diethylamine (diethylamine: acetonitrile=20:80, v/v) into the synthesis column, stand still for 30 minutes, twice, can effectively remove β-nitrile ethyl group, prevent β-nitrile ethyl group The group is combined with the amino group (-NH2) in the process of ammonolysis, and part of the amino group loses its activity, increasing the yield of the product.

Remove MMT protecting group:

Put the synthesis column on the synthesis instrument, and use TCA to remove the MMT protection group. Since the MMT protection group is more difficult to remove than the DMT protection group, you can use TCA to remove it one more time, and then wash it with acetonitrile to remove the CPG in the synthesis column. The powder is taken out, placed in a 2mL centrifuge tube, and the upper and lower sand cores are removed.

Conjugation reaction with activated ester:

Use the activated ester N3-C3-NHS ester (CAS Number: 943858-70-6, FW: 113.12), the structural formula is shown in Figure 10, add 0.1M, 100 μL NaHCO ₃ , PH=8.5 buffer in the centrifuge tube, let the CPG powder Add 3e.q. activated esters to the complete immersion buffer, dissolve the activated esters with anhydrous DMSO or DMF, and store them in a refrigerator at -20°C. During the coupling process, since CPG is granular, it is easy to The sediment at the bottom of the centrifuge tube was placed in a shaker with a constant temperature of 37° C. and a rotation speed of 220 rotations per minute, and the reaction time was 6 hours.

Add 1000 μL of ddH ₂ O to the centrifuge tube, oscillate, and centrifuge at 12,000 rpm for 1 minute to precipitate the CPG at the bottom of the centrifuge tube. Use a pipette to absorb the supernatant and repeat 5 times to remove the unreacted residual ester. To prevent the residual ester from reacting with the 3-terminal amino group during the process of ammonolysis.

Sample analysis:

Dry the sample in a centrifuge, add ddH ₂ O to dissolve, shake, pass through the membrane with a 0.22 μm filter column, filter CPG and particulate matter, transfer to a sample bottle for analysis, HPLC reverse phase analysis: Waters 2695, Waters 2487, Analytical column 4.6*150mm XBridge C18 3.5μm, A mobile phase (100% acetonitrile), B mobile phase (0.05M TEA).

LC-MS molecular weight detection, take 0.2～0.5OD samples for LC-MS molecular weight detection, compare the actual molecular weight with the theoretical molecular weight, if the deviation is within 0.1%, there is no obvious N-peak, the result is judged to be qualified, otherwise it is not qualified.

Example 1

This example tests the influence of different aminolysis conditions on the amide bond in nucleic acid molecules.

Using the test molecule 5'-CY5-TTCTGACCTGAAGGCTCTGCGCG-BHQ2-3', wherein the 5' end is CY5 condensed with the amino group and connected through an amide bond, the test molecule is synthesized by the aforementioned solid-phase synthesis method, and the test molecule is subjected to ammonia solution, mixed with 1000 μL of different ammonia solutions (ammonia water, ammonia gas and tert-butylamine-methanol-ddH ₂ O mixture (1:1:1)) and at different temperatures (25°C, 30°C, 37°C, 50°C, 60°C, 63°C, 65°C and 70°C) for ammonolysis, taking the test molecule without ammonolysis reaction as a control, the MS detection chart is shown in Figure 1, in which the ammonium hydrolysis reaction carried out with ammonia water and ammonia gas The bonds are all destroyed, and the results of the ammonolysis reaction are carried out at different temperatures with tert-butylamine-methanol-ddH ₂ O mixed solution as shown in Table 1, which shows that compared with the traditional ammonolysis solution, the unique ammonolysis solution of the present invention is designed It can effectively protect the amide bond formed by the 5' terminal amino group. In addition, by controlling the ammonolysis temperature, the ammonolysis efficiency can be significantly improved under the condition of effectively protecting the amide bond from being destroyed.

Table 1

Example 2

This example prepares a nucleic acid molecule in which the 5'-terminal amino group is modified and the activated ester (NHS) is conjugated, the 3'-terminal amino group is modified and the 3'-terminal amino group remains active.

Taking the nucleic acid sequence of CTCATCAGTACTGTCTGCAGTACA as an example, use the aforementioned solid-phase synthesis method to carry out solid-phase synthesis, 5' amino modification, deamination protection, activated ester and amino conjugation reaction, ammonolysis pretreatment, and mix the obtained product with 1000 μL of ammonia solution (tert-butylamine-methanol-ddH ₂ O mixture, 1:1:1), ammonolysis at 60°C for 6 hours, liquid chromatography analysis and MS analysis, the target peak in the liquid chromatography is obvious (Figure 12), a small amount of synthesis is in progress Failed and unconjugated impurity peaks with esters, target product: Azide-CTCATCAGTACTGTCTGCAGTACA-NH2 (molecular weight: 7790), detected molecular weight: 7786.8, see Figure 13, deviation is 0.04%, determined to be qualified, the sequence and structure of the final product See Figure 11.

In summary, the present invention ingeniously designs a nucleic acid preparation process capable of double-terminal conjugation of different compounds, so as to not only retain the 3'-terminal amino group, but also carry out a conjugation reaction between the 5'-terminal amino group and the activated ester (NHS). The ammonolysis process realizes the efficient separation of nucleic acid molecules from the solid-phase support and the removal of protective groups without destroying the amide bond, and overcomes the modification of the 3' end with an amino group, and the 5' end amino group needs to be conjugated with an activated ester Reaction, 3' end and 5' end connection of specific compounds will cause conflicts, increase the position of the modification group in the synthetic sequence, provide a new synthesis idea, and promote the application of molecular biology.

The applicant declares that the present invention illustrates the detailed methods of the present invention through the above-mentioned examples, but the present invention is not limited to the above-mentioned detailed methods, that is, it does not mean that the present invention must rely on the above-mentioned detailed methods to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims (10)

1. A method for synthesizing a nucleic acid capable of double-end conjugation to different compounds, the method comprising:

synthesizing nucleic acid molecules from a 3' end to a 5' end by adopting a solid phase synthesis method to obtain the nucleic acid molecules of which the 3' end is connected to a solid phase carrier, connecting amino to the 5' end of the nucleic acid molecules of which the 3' end is connected to the solid phase carrier, mixing the nucleic acid molecules connected with the amino with activated ester for conjugation reaction, mixing the products of the conjugation reaction with aminolysis solution for aminolysis reaction to obtain the nucleic acid of different compounds capable of being conjugated at two ends;

the ammonolysis solution contains tert-butylamine and methanol.

2. The method of claim 1, wherein the solid support comprises microporous glass beads;

preferably, the activated ester comprises a succinimide activated ester and/or an isothiocyanate activated ester;

preferably, the succinimide-activated ester comprises an azido-C3-succinimide ester.

3. The method for synthesizing a nucleic acid capable of double-end conjugation of different compounds according to claim 1 or 2, wherein the ammonolysis solution further comprises water;

preferably, the volume ratio of the tert-butylamine to the methanol to the water in the ammonolysis solution is 1:1 (1-10).

4. The method for synthesizing a nucleic acid capable of double-end conjugation of different compounds according to any one of claims 1 to 3, wherein the conjugation reaction comprises:

mixing the nucleic acid molecules connected with the amino groups with activated ester and an alkaline buffer solution to carry out a conjugation reaction;

preferably, the alkaline buffer comprises a sodium bicarbonate solution;

preferably, the temperature of the conjugation reaction is 30-40 ℃ and the time is 3-10 h.

5. The method for synthesizing nucleic acid capable of double-end conjugation of different compounds according to any one of claims 1 to 4, wherein the temperature of the aminolysis reaction is not higher than 60 ℃;

preferably, the temperature of the ammonolysis reaction is 50 to 60 ℃.

6. The nucleic acid method for synthesizing a nucleic acid capable of double-end conjugation of different compounds according to any one of claims 1 to 5, wherein the ammonolysis reaction further comprises a pretreatment step;

preferably, the pre-treatment comprises:

mixing the product of the conjugation reaction with a diethylamine-acetonitrile mixed solution, and collecting a solid;

preferably, the volume ratio of the diethylamine to the acetonitrile in the mixed solution of diethylamine and acetonitrile is 1 (1-4).

7. The nucleic acid method for the synthesis of a nucleic acid capable of double-end conjugation of different compounds according to any of claims 1 to 6, wherein said amino group has a protecting group;

preferably, the protecting group comprises p-methoxytrityl;

preferably, the conjugation reaction further comprises a step of removing the protecting group on the amino group;

preferably, the method for removing the protecting group on the amino group comprises:

mixing the nucleic acid molecule with the amino group and trichloroacetic acid, the solid was collected.

8. The method for synthesizing a nucleic acid capable of double-end conjugation of different compounds according to any one of claims 1 to 7, comprising the steps of:

(1) Synthesizing nucleic acid molecules from the 3' end to the 5' end by a solid phase synthesis method to obtain nucleic acid molecules with the 3' end connected to a solid phase carrier;

(2) Connecting amino with a protecting group to the 5 'end of the nucleic acid molecule of which the 3' end is connected to the solid phase carrier, mixing the nucleic acid molecule connected with the amino with trichloroacetic acid, collecting a solid, mixing the solid with activated ester, and carrying out a conjugation reaction;

(3) And mixing the product of the conjugation reaction with a mixed solution of diethylamine and acetonitrile, collecting the solid, mixing the solid with an ammonolysis solution, and carrying out ammonolysis reaction to obtain the nucleic acid of different compounds capable of being conjugated at two ends.

9. A nucleic acid capable of double-end conjugation to different compounds, which is prepared by the method for synthesizing a nucleic acid capable of double-end conjugation to different compounds according to any one of claims 1 to 8.

10. Use of a nucleic acid as claimed in claim 9 capable of double-end conjugation to different compounds for the preparation of nucleic acid probes and/or primers.

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