JPS6379897A - Storage of polynucleotide - Google Patents

Abstract

PURPOSE:To enable stable storage of the titled compound in high storage yield, by synthesizing a polynucleotide with a polynucleotide synthesis apparatus by phosphite process, finishing the reaction by capping step and leaving the product in the capping liquid. CONSTITUTION:A polynucleotide is synthesized by phosphite process using a polynucleotide synthesis apparatus constructed to feed the reaction raw materials 7-10, reagents, solvents 2-6, etc., necessary in the polynucleotide synthesis reaction to a reactor 11 with pressure of nitrogen gas supplied from a bomb 1 in prescribed order of feeding controlled by a controlling means 12 and discharge the raw materials, etc., from the reactor. In the above process, the polynucleotide synthesis reaction is finished by the capping step and the produced polynucleotide is stored in the capping liquid used in the capping reaction to enable the stable storage of the polynucleotide.

Description

DETAILED DESCRIPTION OF THE INVENTION Technical Field of the Invention The present invention relates to a method for preserving polynucleotides that are reaction products when polynucleotides are synthesized using a polynucleotide synthesizer.

Technical background of the invention and its problems In recent years, molecular biology represented by genetic engineering has made rapid progress, and along with this, deoxyribonucleic acid (DNA)
), and many attempts are being made to synthesize polynucleotides that constitute small nucleic acids (RNA).

Polynucleotides are either nucleotides in which a phosphoric acid is bound to a deoxylipidic small nucleoside, which is a combination of four types of nucleic acids g, aw, or thymine, adenine, guanine, cytosine, or thymine, and deoxylipose;
The phosphotriester method has traditionally been used to synthesize this polynucleotide. However, since pentavalent phosphoric acid is used in this phosphoric acid triester method, there is a problem in that the activity is low.

For this reason, when synthesizing polynucleotides, the phosphite method using trivalent phosphotriesters, which are rich in reaction activity, has come to be widely adopted. The following four steps (a) to (d) are usually required to synthesize a polynucleotide using the phosphin-I method.

(a) A step in which a trityl group, which is a protecting group at the 5'-position of a nucleoside bonded to a support, is cleaved with an acid to convert the 5-position of the nucleoside into water Ml (deprotecting group step).

(b) Next, this hydroxyl group is reacted with an amidite to which any one of adenine, guanine, cytosine, or thymine is bound, such as N,N-diisopropylphosphoramidite activated with tetrazole or the like, to form a phosphite. process (coupling process).

(C) A step (oxidation step) of oxidizing the phosphite obtained in the camping step using a reagent consisting of iodine, tetrahydrofuran, 2,6-luzidine, water, etc.

(d) A step (capping step) of protecting the 5-position hydroxyl group of the nucleotide that did not react in the coupling step using a capping solution consisting of anhydrous vinegar fJaJ3, dimethylaminopyridine, or the like.

The above four steps may be performed in the order of deprotection step, coupling step, oxidation step, and capping step, but in some cases, the deprotection step, coupling step, capping step, and oxidation step may be performed in the order of deprotection step, coupling step, capping step, and capping step. It may be done in order.

In the polynucleotide synthesis process, which consists of the four steps described above, a washing step using a washing solution such as acetonitrile is usually inserted between each of the above steps, so that each step is performed in an impurity-free state. It is made to proceed with.

Since the synthesis of small nucleotides is too complicated to perform manually, the reaction raw materials, reagents, solvents, etc. necessary for each of the above steps are automatically supplied to the reactor in sequence and then discharged. A polynucleotide synthesizer configured as follows has been proposed.

However, in such a polynucleotide synthesizer, after the polynucleotide synthesis reaction is completed, the reaction system is washed with a washing solution such as acetonitrile, and the resulting polynucleotide and nucleotide oligomers are washed away in the washing solution. It was saved.

However, if the obtained nucleotide oligomer is stored in a washing solution such as acetonitrile, even if the nucleotide oligomer is used again in a reaction, the polynucleotide with an extended chain can be efficiently converted into one polynucleotide. The inventors have found that it is not possible to do so.

The inventors of the present invention conducted extensive research into the reason for this, and found that
The 5-terminus of the nucleotide oligomer is usually held by a trityl group such as dimethoxytrityl, but the trityl group holding the 5-terminus of the nucleotide oligomer can be l111 in washing solvent! It has been found that the resulting polynucleotide oligomers cannot be stably stored. Solving such problems 1
After further investigation, the inventors discovered that it is sufficient to complete the polynucleotide synthesis reaction at a specific step and store the resulting nucleotide oligomer in a specific solution, thereby completing the present invention.

OBJECTS OF THE INVENTION The present invention aims to solve the problems associated with the conventional techniques 1+toi as described above. The purpose of the present invention is to provide a method for preserving polynucleotides in which polynucleotides can be stably preserved at a high preservation rate without being significantly degraded.

Invention 8! ! The method for storing polynucleotides is to supply the reaction raw materials, reagents, solvents, etc. necessary for the polynucleotide synthesis reaction to the reactor in a predetermined order, and then to discharge the polynucleotides into the polynucleotide synthesizer according to the present invention. When storing polynucleotides synthesized by the phosphite method using a polynucleotide synthesizer configured as shown in FIG. The method is characterized in that the obtained polynucleotide is stored in the capping solution used in the capping step.

In the polynucleotide storage method using the polynucleotide synthesizer according to the present invention, the polynucleotide synthesis reaction is completed in a capping step, and the obtained polynucleotide is stored in the capping solution used in the capping solution. Therefore, the removed polynucleotide is
This polynucleotide can be stably stored at a high preservation rate without being significantly degraded.

DETAILED DESCRIPTION OF THE INVENTION The method for preserving polynucleotides in the polynucleotide synthesizer d5 according to the present invention will be specifically explained below.

First, an example of the polynucleotide synthesis apparatus used in the present invention will be described with reference to the drawings. In the drawing, reference numeral 1 is for the inert gas (nitrogen gas), 2 is for the lysate, and 3 to 6 are for the reagents. Bottle, 7-10i, raw material bottle, 11
is a reactor, and 12 is a control means.

The N2 gas filled in the cylinder 1 is passed through the pressure regulating valve 13 and the distributor 14a to the solvent bottle 2 and the reagent bottle 3.
~6, raw materials are sent to bottles 7 to 10, and under the pressure of the N2 gas, the solvent, reagent, and raw materials are sent to the reactor 11 through the channel 14b (consisting of a thin tube of about 1 sφ). In addition, N2 gas passes through the orifice 15 and the flow path 14b to the reactor 1.
Sent to 1. The orifice 15 serves to limit the flow rate of the N2 gas to prevent overflow when bubbling the N2 gas inside the reactor 11. N2
The gas is normally used after being dried with a desiccant such as molecular sieves and filtered with a filter. In addition to N2 gas, helium, argon, etc. can be used as the inert gas.

Solvent bottle 2 contains a solvent such as acetonitrile, reagent bottle 3
Deprotection bases such as trichloroacetic acid and dichloroacetic acid are used for
Reagent bottle 4 contains iodine, tetradidrofuran, 2,6-
Reagent bottle 5 is filled with an oxidizing agent such as Luzidine and water, a capping agent such as a mixture of acetic anhydride and dimethylaminopyridine, and reagent bottle 6 is filled with an activating agent such as tetrazole, tetrahydrofuran, acetonitrile, etc. has been done. In addition, adenine (A),
Phosphoramidites (nucleotide reagents) containing guanine (G), cytosine (C) and thymine (T> as bases)
It is filled with a solution consisting of and a solvent.

Check valves 16. are provided on the inlet sides of the reagent bottles 3, 4.5, respectively.
17 and 18 are provided to prevent vapors and mist of the deprotecting base, oxidizing agent, and capping agent from flowing back through the channel 14b and into other bottles.

A solenoid valve 19 is provided in the flow path '14b, and the flow path '14b is also provided with a solenoid valve 19.
Solenoid valves 20 to 2 are installed on the outlet sides of the bottles 2 to 10, respectively.
8 is provided, and electromagnetic valves 29 to 32 are provided on the bottom side and the top side of the reactor 11 in the flow path 14b, respectively. These electromagnetic valves 19 to 32 are controlled by a control means (programmable controller) 12 to open and close.

The solenoid valve 19 is a two-way valve that listens when energized, and the solenoid valve 20 ~
Numeral 30 is a three-way valve that communicates with either the dotted line or the solid line when energized, and the solenoid valves 31 and 32 are three-way valves that switch from the solid line state to the dotted line state when energized.

Next, a general method of operating such a polynucleotide synthesizer will be explained. In the reaction operation, first, a porous glass having, for example, thymine nucleoside bonded thereon is supported in the reactor 11. When filling the body (support) and then operating the control means 12, the restraining means 12
Solenoid valves 19-3 according to the contents of the program specified in 62.
2 are sequentially opened and closed to perform the line barge process → cleaning culm → four deprotection processes → washing) 1) process → cutting rod → cleaning process → oxidation process - cleaning process → capping process → washing) p process.

The above is necessary for IH of a polynucleotide synthesizer.

In the present invention, polynucleotides are synthesized by the phosphite method using such a polynucleotide synthesizer,
When storing the obtained nucleotide oligomer in this polynucleotide synthesizer, the polynucleotide synthesis reaction is completed in a capping step, and the nucleotide oligomer (polynucleotide) thus obtained is stored in a capping solution.

As the capping liquid used at this time, acid anhydrides such as acetic anhydride, phenyl isocyanate, diethyl monotriazolophosphite, etc. are used, and if necessary, N,N-dimethylaminopyridine, N,N-diethylaminopyridine, etc. are used. , N,N-dimethylaminopyridine can be used in combination with a base such as N,N-dimethylaminopyridine.The capping liquid can be a mixture of an acid anhydride such as acetic anhydride and a base such as N,N-dimethylaminopyridine. is particularly preferably used.

When a polynucleotide synthesized using a polynucleotide synthesizer is stored in a capping solution, the five retaining groups such as the dimethoxytrityl group that retains the 5' end of the obtained polynucleotide are stored in the capping solution. Therefore, it is possible to preserve the obtained polynucleotide with a high preservation rate.

Although the above explanation has been made using deoxyribonucleic acid as an example for convenience, it goes without saying that the polynucleotide of the present invention also includes other nucleic acids such as ribonucleic acid.

Effects of the Invention In the method for preserving polynucleotide in the polynucleotide synthesizer according to the present invention, the polynucleotide synthesis reaction is completed in the capping step, and the polynucleotide synthesis reaction is completed in the capping step. What is your polynucleotide? Since the 1d polynucleotide is stored in the capping solution used in the above, it can be stably stored at a high preservation rate without much decomposition of the polynucleotide.

The present invention will be explained below with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Using a polynucleotide synthesizer as shown in the drawing 1,
A polynucleotide consisting of 30 consecutive nucleotides was synthesized.

At this time, the synthesis reaction of polyscleotide is completed in a capping step, and the polyscleotide whose 5-terminus is protected with a dimethoxytrityl group is placed in a capping solution consisting of a mixture of acetic anhydride and N,N-dimethylaminopyridine at room temperature. Saved with.

After storage for 1 hour, after storage for 5 hours, and after storage for 24 hours,
Each 1q polynucleotide was post-treated (deprotected) by a predetermined method, and the product was analyzed by high performance liquid chromatography to determine the proportion of polyscleotide with dimethoxytrityl groups and the retention rate.

The results are shown in Table 1.

Comparative Example 1 J3 in Example 1 carried out the synthesis reaction of polyscleotide.
The reaction was completed in the same manner as in Example 1, except that the capping step was completed, and then the reaction system was washed with acetonitrile, and the obtained polyscleotide was stored as it was in acetonitrile.

The results are shown in Table 1.

Comparative Example 2 In Example 1, the polyscleotide synthesis reaction was completed in the capping step, and then the reaction system was washed with acetonitrile, and after removing acetonitrile,
Example 1 except that the obtained polynucleotide was stored.
I did the same thing.

The results are shown in Table 1.

Table 1 Table 1 shows that polynucleotides can be preserved at a high preservation rate by terminating the polynucleotide synthesis reaction in the capping step and storing the resulting polynucleotide in the capping solution used in the capping step.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram of a polynucleotide synthesis apparatus used in the present invention. 1...Cylinder 2-6...Solvent, reagent bottle 7-
10... Raw material bottle 11... Reactor 12... Control means

Claims (1)

[Claims] 1) Synthesized by the phosphite method using a polynucleotide synthesizer configured to supply reaction materials, reagents, solvents, etc. necessary for polynucleotide synthesis reactions into a reactor in a predetermined order and then discharge them. When storing polynucleotides in a polynucleotide synthesizer, the polynucleotide synthesis reaction is terminated in a capping step,
A method for preserving polynucleotides in a polynucleotide synthesizer, comprising preserving the obtained polynucleotides in a capping solution used in a capping step.