WO2019158007A1

WO2019158007A1 - Method and system for synthesizing oligonucleotide

Info

Publication number: WO2019158007A1
Application number: PCT/CN2019/074588
Authority: WO
Inventors: 王勇; 陈世宏; 周超; 孙宝策; 冯利鹤; 黄小罗; 沈玥; 李汉东; 章文蔚; 徐讯
Original assignee: 深圳华大生命科学研究院
Priority date: 2018-02-13
Filing date: 2019-02-02
Publication date: 2019-08-22
Also published as: CN111479818A

Abstract

Provided is a method and system for synthesizing a nucleic acid with "a synthesis pool". The method is based on phosphoramidite solid-phase synthesis of a nucleic acid in a four-step cycle, wherein deprotection, coupling, capping and oxidation are performed in a first reaction vessel, a second reaction vessel, a third reaction vessel, and a fourth reaction vessel that are independent of one another. The method relates to using a multi-base nucleotide as a nucleic acid synthesis monomer, such that a nucleic acid having a longer chain can be rapidly synthesized, the synthesis error rate is low, and a reagent can be repeatedly used so that the synthesis efficiency is greatly improved.

Description

Method and system for synthesizing oligonucleotide

Technical field

The invention relates to the field of nucleic acid synthesis.

Background of the invention

Nucleic acid is the basic genetic material in life. In vitro artificial synthesis of nucleic acids can replicate any naturally occurring nucleic acid function or create new nucleic acid functions, depending on the needs of the research and application. With the development of genomics, molecular biology, systems biology, bioinformatics and synthetic biology, synthetic nucleic acids have a wide range of applications in cell engineering, gene editing, disease diagnosis and treatment, and new material development. value.

Since Todd, the Khoran team in the 1950s, for the first time since the publication of nucleic acid synthesis, its synthesis has undergone long-term development. The current classical methods include column synthesis developed in the 1980s and development in the 1990s. High-throughput synthesis based on microarrays. These methods are synthesized in units of single deoxynucleotides. The synthesis principle is mostly based on four-step cyclic solid phase synthesis of phosphoramidite chemistry: including: deprotection, coupling, capping, oxidation. Due to the incompleteness of each step reaction and the accompanying possible side reactions (such as deadenosine) and the decrease in the concentration of the reactants, the error rate of nucleic acid synthesis increases sharply with the elongation of the single strand of the nucleic acid, and the yield sharply decreases. Based on this single-base synthesis of phosphoramidite chemistry, some mature progress has been made, and major commercial companies have also launched corresponding primer synthesis services. However, due to the lack of technological breakthroughs in leaps and bounds, at present, the mature primer synthesis services of major companies are only about 100 bp, and the single base error rate is also around 1/200. Although some literatures and a few commercial companies have reported that they can synthesize primers with longer lengths and lower error rates, their practical applications are often not widely used in synthetic experiments or external services due to their poor stability. . In addition, existing single-base nucleic acid synthesis methods, including chemical synthesis, electrochemical synthesis, and photochemical synthesis of porous glass, are based on a four-step method of chemical synthesis of phosphoramidite, and nucleic acid monomers are sequentially added one by one according to a predetermined sequence. Based on single-base nucleic acid synthesis methods, some commercial synthesizers have entered the market. These synthesizers can be broadly divided into two column synthesizers and microarray synthesizers. A column synthesizer, such as Dr. oligo 192, is a solid phase synthesis reaction on a porous reaction column of the order of centimeters by the addition of a solenoid valve control reagent. The method has a low error rate, but the synthesis flux is not high. There is also more material required. Microarray synthesizers, such as the CustomArray synthesizer, reduce the synthesis reaction to micron-sized reaction wells, with tens of thousands of reaction wells on a single chip, which reduces feedstock consumption to a certain extent while increasing synthesis throughput. However, the yield is low, the electrochemical reaction is not easy to control, and the error rate is high. In addition, from the point of view of the injection method, the column and microarray nucleic acid synthesizers are all added to the synthesis column or the synthetic chip through the pre-laid pipeline, and the added reagent is greatly excessive, which causes the reagent to be extremely Great waste and low material usage.

In summary, prior to the present invention, due to the incompleteness of each step reaction and the accompanying possible side reactions, the error rate of the single-stranded nucleic acid synthesized by the common single-base nucleic acid synthesis method rapidly increases with the increase of the length, correspondingly The yield is also drastically reduced, which results in a significant limitation on the length and yield of the nucleic acid synthesis product. At present, the commercial column synthesizer has low synthesis flux and low material utilization rate, and cannot meet the demand for large-scale low-cost nucleic acid synthesis in the future. In contrast, although microarray chips enable high-throughput nucleic acid synthesis, the yield of this synthesis method is small, the error rate is high, and the product is difficult to separate from the mixture, increasing the cost of subsequent operations, such as polymerase chain reaction and Gene assembly operations. In addition, both column and microarray nucleic acid synthesizers require reagent input and output lines, and the amount of reagents used in the synthesis process is large, which greatly increases the cost of synthesis.

Therefore, from the perspective of commercialization of low cost, high efficiency, low error rate and long base, the existing nucleic acid synthesis technology needs further improvement and optimization.

Summary of the invention

In view of the problems in the above nucleic acid synthesis, including the limitation of direct synthesis length, low synthesis efficiency and relatively high cost, the present invention proposes a novel method for coping with these problems, namely a "synthesis pool" nucleic acid synthesis method. More particularly, the present invention relates to synthetic pool multi-base synthesis methods (e.g., two-base nucleic acid synthesis, three-base nucleic acid synthesis, four-base nucleic acid synthesis, etc.). Compared with the existing single-base synthesis method, as long as the corresponding multi-base nucleotide is synthesized in advance as a nucleic acid synthesis monomer under the same number of synthesis cycles, the "synthesis pool" nucleic acid synthesis method of the present invention is used. The nucleic acid single chain with longer chain length can be obtained quickly, and the synthesis efficiency can be greatly improved by the synthesis strategy, the synthetic chain length can be greatly extended correspondingly, and the synthesis error rate can be correspondingly greatly reduced.

Thus, in one aspect, the invention provides a method of synthesizing an oligonucleotide, the method comprising:

a) providing a solid phase carrier represented by formula (I) or a stereoisomer of formula (I),

among them:

Nu is a single bond or

Wherein X ^{2 in} Nu is attached to R ¹³ ;

X ^{1 is} independently -O- or -S-;

X ^{2 is} independently -O-, -S- or -NR-;

X ^{3 is} independently -O-, -S-, -CH ₂ - or -(CH ₂ ) ₂ -;

X ^{4 is} independently =O or =S;

R ¹ is a protecting group;

R ^{2 is} independently -H, -F, -NHR ⁶ , -CH ₂ R ⁶ or -OR ⁶ ;

R ^{3 is} independently -OCH ₂ CH ₂ CN, -SCH ₂ CH ₂ CN, a substituted or unsubstituted aliphatic group, -OR ⁷ or -SR ⁷ ;

R is -H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or an amine protecting group;

R ⁶ is -H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, or a protecting group;

R ⁷ is a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group;

Each B is independently a base that is modified or unmodified;

R ¹³ is a solid phase carrier or -Y ¹ -LY ¹ -R ¹⁴ ;

Y ¹ is a single bond, a double bond, -C(O)-, -C(O)NR ¹⁷ , -C(O)O-, -NR ¹⁷ - or -O-;

L is a single bond, a double bond, a substituted or unsubstituted aliphatic group, or a substituted or unsubstituted aryl group;

R ¹⁷ is -H, a substituted or unsubstituted aliphatic group, or a substituted or unsubstituted aryl group;

R ¹⁴ is a solid phase carrier; and

q is 0 or a positive integer;

b) contacting the solid support with a deprotecting agent in a first reaction vessel containing a deprotecting agent to form a deprotected solid support represented by formula (II) or a stereoisomer of formula (II) ,

Wherein X ⁵ is -OH or -SH; the other groups are as defined above;

c) subjecting the solid support to a phosphoramidite activator and a sub-reagent in a second reaction vessel comprising a phosphoramidite activator and a phosphoramidite monomer or multimer or a stereoisomer thereof of formula (III) Phosphoramide monomer or multimer or a stereoisomer thereof,

among them:

R ⁴ and R ⁵ are each independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl group;

R ⁴ and R ⁵ together with the nitrogen to which they are bonded form a heterocycloalkyl or heteroaryl group, wherein the heterocycloalkyl or heteroaryl group is preferably a five or six membered ring;

n is 0 or a positive integer;

Other groups are as defined above;

Wherein the phosphoramidite monomer or polymer represented by the formula (III) or a stereoisomer thereof is coupled with the deprotected solid phase carrier or stereoisomer represented by the formula (II), thereby forming a formula ( a first intermediate or a stereoisomer thereof as shown in IV),

d) optionally, contacting the solid support with a blocking agent in a third reaction vessel comprising a blocking agent, wherein formula (II) is not reacted with the phosphoramidite monomer or multimer in step c) The deprotected solid support or the X ⁵ group of the stereoisomer is blocked;

e) contacting the solid support with an oxidizing agent or a vulcanizing agent in a fourth reaction vessel containing an oxidizing agent or a vulcanizing agent, wherein the trivalent phosphorus group in the first intermediate is oxidized or vulcanized to form the first formula (V) a second intermediate or a stereoisomer thereof;

Where r is a positive integer;

Wherein the first reaction vessel, the second reaction vessel, the third reaction vessel and the fourth reaction vessel are independent of each other;

f) optionally repeating steps b), c), e) or b) to e) one or more times, wherein the final step is step b), d) or e), thereby synthesizing the desired oligonucleotide .

In an embodiment of the present invention, if the sequence of the oligonucleotide to be synthesized has a short chain length (for example, the number of cycles is ≤ 25), the capping step may be omitted if the sequence of the sequence of the oligonucleotide to be synthesized is If the length is long (for example, the number of cycles > 25), the capping step is usually not omitted.

In a preferred embodiment, a washing step is also included between steps b and c and between steps d and e. Preferably, the solid phase carrier is washed by contacting the solid phase carrier with a washing reagent in a fifth reaction vessel containing a detergent, wherein the fifth reaction vessel is combined with the first reaction vessel, the second reaction vessel, the third reaction vessel, The fourth reaction vessels are independent of each other. In a specific embodiment, the detergent can be acetonitrile.

In a preferred embodiment, in step f), each time steps b), c), e) or b) to e) are repeated, the deprotecting agent in the first reaction vessel, in the second reaction vessel The activator is reused with the phosphoramidite monomer or multimer or stereoisomer thereof, the capping agent in the third reaction vessel, and/or the oxidizing agent or vulcanizing agent in the fourth reaction vessel. The phrase "reuse" means that the reagents required for nucleic acid synthesis, such as deprotecting agents, activators and phosphoramidite monomers or polymers or stereoisomers thereof, blocking agents, oxidizing agents or vulcanizing agents, etc. Separately placed in a plurality of reaction vessels independent of each other, the reagents are not discarded after being contacted with the solid phase carrier and reacted once, but are again contacted and reacted with the solid phase carrier in one or more subsequent steps. Thereby, the reaction reagent contained in the reaction vessel is used multiple times. For example, in the method of the present invention, when the step b) is carried out for the first time, the solid phase carrier is contacted with the deprotecting agent in the first reaction vessel containing the deprotecting agent, and the deprotecting agent is not after the reaction is completed. Will be discarded and still remain in the first reaction vessel. Thereafter in step f), step b) is repeated one or more times. When step b) is repeatedly carried out for the first time, the deprotecting agent remaining in the first reaction vessel is used for the second time in contact with the solid phase carrier, and likewise, the deprotecting agent is not discarded after the reaction is completed. Retained in the first reaction vessel. When step b) is repeated a second time, the deprotecting agent remaining in the first reaction vessel is used for the third time in contact with the solid support. By analogy, the number of repetitions required is ultimately determined based on the length of the oligonucleotide to be synthesized. This achieves repeated use of the deprotecting agent contained in the first reaction vessel.

In a preferred embodiment, n may be selected from a positive integer of 0, 1, 2, 3, 4, 5, 6, 7, or more. When n = 0, the method as described above is equivalent to the prior art single base nucleic acid synthesis method. Thus, in a preferred embodiment, n is a positive integer greater than or equal to one. In a preferred embodiment, n is 1, 2 or 3. Preferably, n is 1.

In a preferred embodiment, X ¹ is -O-; X ² is -O-; X ³ is -O-; X ⁴ is =O; X ⁵ is -OH.

In a preferred embodiment, the phosphoramidite activator is selected from the group consisting of tetrazole, S-ethylthiotetrazole, dicyanoimidazole or pyridinium salt.

In a preferred embodiment, the oxidizing agent is selected from the group consisting of iodine solutions.

In a preferred embodiment, the vulcanizing agent is selected from the group consisting of 3-amino-[1,2,4]-dithiazole-5-thione or 3H-benzodithiazol-3-one 1,1-dioxide.

In a preferred embodiment, the deprotecting agent is selected from the group consisting of a solution of trichloroacetic acid in dichloromethane or a solution of trifluoroacetic acid in acetonitrile.

In a preferred embodiment, the phosphoramidite monomer or multimer represented by formula (III) or a stereoisomer thereof may be a phosphoramidite monomer or polymer as shown in formula (VI) or Its stereoisomers:

In Structural Formula VI, the definitions of B and R ² are the same as in Formula I. R ⁸ is a substituted or unsubstituted trityl group such as 4,4'-dimethoxytrityl. R ¹⁰ and R ¹¹ are each independently a substituted or unsubstituted aliphatic group. R ¹⁰ and R ^{11 are} preferably an isopropyl group. m is 0, 1, or 2.

In a preferred embodiment, R ¹ is an acid labile protecting group or a trialkylsilyl group, e.g. tert-butyldimethylsilyl or triisopropylsilyl silyl group. In a preferred embodiment, R ¹ is substituted or unsubstituted trityl, 9-(phenyl)xanthene (also known as "pixyl") or tetrahydropyranyl (also known as "THP""). In a more preferred embodiment, R ¹ is unsubstituted trityl, monoalkoxytrityl, dialkoxytrityl, trialkoxytrityl, THP or 9 -Phenylindole. Most preferably, R ¹ is 4,4'-dimethoxytrityl (also known as "DMT").

In one embodiment, R ² represents C-allyl. In a preferred embodiment, R ² is -H, -O or -OCH ₂ CH ₂ OMe.

In a preferred embodiment, R ^{3 is} independently -OCH ₂ CH ₂ CN, -SCH ₂ CH ₂ CN, 4-cyanobut-2-enylthio, 4-cyanobut-2-enyloxy, Allylthio, allyloxy, 2-butenylthio or 2-butenyloxy. In a preferred embodiment, R ³ is -OCH ₂ CH ₂ CN or -SCH ₂ CH ₂ CN.

In a preferred embodiment, the method further comprises a treatment with a base synthetic oligonucleotides to remove from -OCH _₂ CH ₂ CN or -SCH _₂ CH ₂ CN in -CH _₂ CH ₂ CN.

In a preferred embodiment, each of R ⁴ and R ⁵ is isopropyl.

In a preferred embodiment, R ⁷ is o-chlorophenyl or p-chlorophenyl.

In a particular embodiment, B may also be H, for example, when one or more abasic moieties are present.

In a particular embodiment, the phosphoramidite monomer or multimer or a stereoisomer thereof of formula (III) is selected from one of the following 20 compounds or a stereoisomer thereof:

Wherein Bz is a benzoyl group and ib is an isobutyryl group.

As described herein above, the solid support can be any solid support suitable for solid phase oligonucleotide synthesis, such as, but not limited to, pore size controllable glass spheres (also known as "CPG"), polystyrene, microporous polyamides. For example, polydimethylacrylamide, polyethylene glycol coated polystyrene, and polyethylene glycol supported on polystyrene, such as those sold under the tradename Tentagel.

Preferably, the solid support is a CPG bearing an amino-modified reactive functional group. The particle diameter of the CPG may be less than or equal to 5 μm, less than or equal to 25 μm, less than or equal to 50 μm, less than or equal to 100 μm, less than or equal to 200 μm, less than or equal to 500 μm or more; and the pore diameter may be less than or equal to

less than or equal to

Or bigger. The linking molecule of the modified solid phase carrier may have an ester group, a lipid group, a thioester group, an o-nitrobenzyl group, a coumarin group, a hydroxyl group, a thiol group, an anthracene ether group, a carboxyl group, an aldehyde group, an amino group, an amine group, A compound of any one or more of an amide group, an alkenyl group, or an alkynyl group.

The aliphatic group used in the present invention includes a linear or branched C ₁ -C ₁₈ hydrocarbon group which is fully saturated or contains one or more non-aromatic double bonds, or is fully saturated or contains one or more non-conjugated double A C ₃ -C ₁₈ cyclic hydrocarbon group of the bond. The lower alkyl group is a fully saturated linear or branched C ₁ -C ₈ hydrocarbon group or a C ₃ -C ₈ cyclic hydrocarbon group. The aliphatic group is preferably a lower alkyl group.

The aromatic groups used in the present invention include carbocyclic aromatic ring systems (e.g., phenyl) and carbocyclic aromatic ring systems fused to one or more carbocyclic aromatic or non-aromatic rings (e.g., naphthyl, anthracenyl, and 1,2,3,4-tetrahydronaphthyl).

As used herein, heteroaryl groups include heteroaryl ring systems (eg, thienyl, pyridyl, pyrazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, oxazolyl, furyl, pyrrolyl, Imidazolyl, pyrazolyl, triazolyl, pyrimidinyl, pyrazinyl, thiazolyl, isoxazolyl, isothiazolyl, tetrazolyl or oxadiazolyl) and one of the carbocyclic aromatic rings, carbocyclic non- A heteroaromatic ring system in which an aromatic ring, heteroaryl ring or heterocycloalkyl ring is fused to one or more other heteroaryl rings (eg, benzothienyl, benzimidazole, indole, tetrahydroanthracene, Azaindole, carbazole, quinoline, imidazopyridine, indole, pyrrolo[2,3-d]pyrimidine and pyrazolo[3,4-d]pyrimidine).

As used herein, an aralkyl group is an aromatic substituent attached to a moiety through an aliphatic group preferably having from 1 to about 6 carbon atoms.

The heterocycloalkyl group as used herein is a non-aromatic ring system which preferably has 5 to 6 atoms and includes at least one hetero atom such as nitrogen, oxygen or sulfur. Examples of heterocycloalkyl groups include morpholine, piperidine, piperazine and the like.

Suitable substituents for aliphatic groups, aromatic groups, aralkyl groups, heteroaryl groups and heterocycloalkyl groups include aryl groups, halogenated aryl groups, lower alkyl groups, halogenated lower alkyl groups (e.g., trifluoromethyl and Trichloromethyl), -O-(aliphatic or substituted aliphatic), -O-(aryl or substituted aryl), benzyl, substituted benzyl, halogen, cyano, nitro, - S-(aliphatic or substituted aliphatic) and -S-(aryl or substituted aryl).

Amine, alcohol and thiol protecting groups are known to those skilled in the art. For an example of an amine protecting group, see Greene et al., Protective Groups in Organic Synthesis (1991), John Wiley & Sons, Inc. (hereinafter referred to as "the book") 309-405 The pages are hereby incorporated by reference in its entirety. Preferably, the amine is protected as an amide or carbamate. See pages 10-142 of this book for examples of alcohol protecting groups, the contents of which are hereby incorporated by reference in its entirety. A preferred alcohol protecting group is tert-butyldimethylsilyl. For examples of thiol protecting groups, see pages 277-308 of the book, the contents of which are incorporated herein by reference in its entirety.

The acid labile protecting group is a protecting group which can be removed by contact with a Bronsted acid or a Lewis acid. Acid labile protecting groups are known to those skilled in the art. Examples of common acid labile protecting groups include substituted or unsubstituted trityl groups (pages 60-62 of the book), substituted or unsubstituted tetrahydropyranyl groups (pages 31-34 of the book). , substituted or unsubstituted tetrahydrofuranyl (pages 36-37 of the book) or 9-phenylxanthene (p. 65 of the book). A preferred acid labile protecting group is a substituted or unsubstituted trityl group such as 4,4'-dimethoxytrityl (also referred to as "DMT"). The trityl group is preferably substituted by an electron donating group such as an alkoxy group.

Nucleoside bases include naturally occurring bases such as adenine, guanine, cytosine, thymine, and uracil, as well as modified bases such as 7-deazaguanine, 7-deaza-8-aza Guanine, 5-propynylcytosine, 5-propynyluracil, 7-deaza adenine, 7-deaza-8-azadenine, 7-deaza-6-oxopurine, 6 -oxopurine, 3-deaza adenosine, 2-nitro-5-methylpyrimidine, 2-oxo-4-methylthio-5-methylpyrimidine, 2-thiocarbonyl-4-oxo- 5-methylpyrimidine, 4-oxo-5-methylpyrimidine, 2-aminopurine, 5-fluorouracil, 2,6-diaminopurine, 8-aminopurine, 4-triazole-5-methylthymidine And 4-triazole-5-methyluracil.

A protected nucleobase is a nucleobase in which the active functional group of the base is protected. Typically, nucleobases have an amine group that can be protected with an amine protecting group, such as by formation of an amide or carbamate. For example, the amine groups of adenine and cytosine are typically protected with a benzoyl protecting group, while the amine groups of guanine are typically protected with isobutyryl, acetyl or t-butylphenoxyacetyl groups. However, other protection schemes can also be used. For example, for rapid deprotection, the adenine and guanine amine groups are protected with a phenoxyacetyl group, while the cytosine amine group is protected with an isobutyryl group. When a multimeric synthetic oligonucleotide of the invention having a protected nucleotide base is used, the conditions for removal of the protecting group will depend on the protecting group employed. When an amino group protected with an amide group form is used, the oligonucleotide may be treated with an alkali solution such as a concentrated aqueous ammonia solution, a normal methylamine solution or a t-butylamine/ammonium hydroxide solution to remove it.

Structural formulae as referred to herein are understood to include the corresponding stereoisomers where appropriate.

In a particular embodiment, the deprotecting agent used in step b) depends on the R ¹ group used. If R ¹ is an acid labile protecting group, the deprotecting agent is selected from the group consisting of acids. If R ¹ is a trialkylsilyl group such as tert-butyldimethylsilyl or triisopropylsilyl, the second intermediate can be treated with fluoride ions to remove R ¹ . Typically, tert-butyldimethylsilyl and triisopropylsilyl groups are removed by treatment with a solution of tetrabutylammonium fluoride in THF. Pages 77-83 a method of removing tert-butyldimethylsilyl protecting groups can be (Protective Groups in Organic Synthesis, 1991 , John Wiley & Sons.Inc.) In Organic Synthesis Greene et al found in the book This reference is incorporated herein by reference in its entirety.

In a specific embodiment, when it is desired to obtain a 5'-terminal protected oligonucleotide therein, if a capping step is performed, the final step of the reaction cycle may be the capping step; if no capping is performed In the step, the final step of the reaction may be an oxidation or sulfurization step. Alternatively, if desired to give the 5'-oligonucleotide without a protective group, the final step of the reaction cycle can be removal of R ^1.

In a specific embodiment, the synthetic oligonucleotide can be an oligoribonucleotide. In a specific embodiment, the synthetic oligonucleotide can be an oligodeoxyribonucleotide.

In a specific embodiment, the synthetic oligonucleotide can be a phosphate ester and thus has only a phosphate linkage (ie, the internucleotide phosphorus is only bonded to oxygen). In another embodiment, the synthetic oligonucleotide may be a phosphorothioate, thus having only a phosphorothioate linkage (phosphor between each nucleotide is bonded to at least one S, preferably only one S) . In yet another embodiment, the synthetic oligonucleotide can be a chimeric oligonucleotide that contains both a phosphate and a phosphorothioate internucleotide linkage.

In the context of the present disclosure, each of the reaction vessels in steps b to e above is collectively referred to as a "synthesis cell", a solid phase carrier is referred to as a "synthetic needle", and a plurality of solid phase carriers are referred to as "synthetic needle sets". Thus, the nucleic acid synthesis method of the present invention is also referred to as a "synthetic pool" nucleic acid synthesis method.

The innovative "synthetic pool" nucleic acid synthesis method of the present invention can realize large-scale control by precisely controlling the reaction time of "synthesis needle" immersed in the "synthesis tank" and the number of cycles of "synthesis pool" under the premise of satisfying the yield. Direct synthesis of long-chain nucleic acid fragments at low cost, high efficiency, and low error rate. The synthesis method can greatly reduce the synthesis cost by recycling the materials in the "synthesis cell", and can also ensure the low error rate of the synthesized product by solid phase synthesis. In addition, the method does not require reagent input and output pipelines, which saves pipeline cost and reduces design complexity. The "synthetic pool" method can avoid cross-contamination and further ensure the accuracy of nucleic acid synthesis; solid phase synthesis "synthesis The Needle" group is scalable and easy to integrate, allowing flexible adjustment of throughput and throughput as needed, and recycling to reduce costs. In summary, the nucleic acid synthesis method based on "synthesis pool" proposed by the invention improves the synthesis efficiency, and also avoids the shortcomings of the current commercial synthesizer, and provides a higher development for the future commercial synthesizer. A new way of feasibility.

More particularly, the "synthetic pool" nucleic acid synthesis method of the present invention is well suited for use in high throughput nucleic acid synthesis, i.e., simultaneous synthesis of a variety of desired nucleic acids.

Accordingly, in a preferred embodiment, the invention provides a method of synthesizing an oligonucleotide, the method comprising:

a) providing a plurality of solid phase supports of formula (I) (which may be the same or different and may be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200 or more) or a stereoisomer of the formula (I),

among them:

Nu is a single bond or

Wherein X ^{2 in} Nu is attached to R ¹³ ;

X ^{1 is} independently -O- or -S-;

X ^{2 is} independently -O-, -S- or -NR-;

X ^{3 is} independently -O-, -S-, -CH ₂ - or -(CH ₂ ) ₂ -;

X ^{4 is} independently =O or =S;

R ¹ is a protecting group;

R ^{2 is} independently -H, -F, -NHR ⁶ , -CH ₂ R ⁶ or -OR ⁶ ;

Each B is independently a base that is modified or unmodified;

R ¹³ is a solid phase carrier or -Y ¹ -LY ¹ -R ¹⁴ ;

R ¹⁴ is a solid phase carrier; and

q is 0 or a positive integer;

b) according to the desired oligonucleotide sequence to be synthesized on each solid phase carrier, one or more of the plurality of solid phase carriers in the first reaction vessel containing the deprotecting agent and the deprotecting agent Contacting to form a deprotected solid phase carrier represented by formula (II) or a stereoisomer of formula (II), and other solid phase carriers may, for example, be placed in an empty reaction vessel containing no deprotecting agent,

Wherein X ⁵ is -OH or -SH; the other groups are as defined above;

c) subjecting said plurality of solid phase supports to a phosphoramidite in a second reaction vessel comprising a phosphoramidite activator and a phosphoramidite monomer or polymer of formula (III) or a stereoisomer thereof The activator is contacted with a phosphoramidite monomer or multimer or a stereoisomer thereof,

among them:

n is 0 or a positive integer;

Other groups are as defined above;

Wherein the phosphoramidite monomer or multimer represented by formula (III) or a stereoisomer thereof is coupled with a deprotected solid phase carrier or stereoisomer represented by formula (II), thereby forming a formula a first intermediate or a stereoisomer thereof as shown in (IV),

d) optionally, contacting said plurality of solid phase supports with a blocking agent in a third reaction vessel comprising a blocking agent, wherein said step c) is not reacted with a phosphoramidite monomer or multimer The deprotected solid phase support represented by formula (II) or the X ⁵ group of the stereoisomer is blocked;

e) contacting the plurality of solid phase supports with an oxidizing agent or a vulcanizing agent in a fourth reaction vessel comprising an oxidizing agent or a vulcanizing agent, wherein the trivalent phosphorus group in the first intermediate is oxidized or vulcanized to form formula (V) a second intermediate or a stereoisomer thereof as shown;

Where r is a positive integer;

In another aspect, the invention also provides a system for nucleic acid synthesis comprising:

One or more solid phase carriers as shown in formula (I) or stereoisomers represented by formula (I),

One or more reaction vessels containing a deprotecting agent,

One or more reaction vessels comprising a phosphoramidite activator and a phosphoramidite monomer or multimer represented by formula (III) or a stereoisomer thereof,

Optionally one or more reaction vessels comprising a capping agent,

One or more reaction vessels containing an oxidizing agent or a vulcanizing agent,

a mobile device for moving the one or more solid phase carriers between reaction vessels, and

A control system for controlling movement of the mobile device.

In a preferred embodiment, the system further comprises one or more reaction vessels comprising a detergent.

In a preferred embodiment, the mobile device controls the one or more solid phase carriers to move simultaneously between the respective reaction vessels.

In a preferred embodiment, the one or more solid phase carriers are detachably secured to the mobile device.

In a preferred embodiment, the mobile device is a robotic arm.

In a preferred embodiment, the control system is a computer operated programmatic control system.

In another aspect, the invention provides a system for nucleic acid synthesis comprising:

a reaction vessel for receiving a deprotection reagent having one or more separate tanks, and the number of said tanks being at least equal to the number of said solid phase supports,

Optionally one or more reaction vessels comprising a capping agent,

A control system for controlling movement of the mobile device.

In a preferred embodiment, the one or more separate tanks each correspond to a solid support.

In a preferred embodiment, the system further comprises an injection device for adding a deprotection reagent to the tank.

The infusion device as described herein can be any device suitable for adding a deprotecting agent to the tank. For example, the infusion device can be an injector containing a deprotecting reagent, such as a syringe. As another example, the injection device can be a reaction vessel containing a deprotection reagent in fluid communication with the tank. Preferably, such an injection device further comprises means for controlling the flow of deprotection reagent into the tank. The means for controlling the flow of deprotection reagent into the tank may include, for example, a valve. The valve's switch can be controlled by the control system. The means for controlling the flow of the deprotection reagent into the tank may also include, for example, a pressure control system or the like. For example, a positive pressure can be created in the injection device such that the deprotection reagent flows from the injection device into the tank.

In a preferred embodiment, the system further comprises a drain for discharging the deprotection reagent from the tank.

The venting device as described herein can be any device suitable for expelling the deprotecting agent from the tank. For example, the discharge device can be a pipette, such as a vacuum aspirator. As another example, the discharge means can be a fluid passage in fluid communication with the tank. Preferably, such a discharge device further comprises means for controlling the flow of deprotection reagent out of the tank. The means for controlling the deprotection reagent to flow out of the tank may comprise, for example, a valve. The valve's switch can be controlled by the control system. The means for controlling the deprotection reagent to flow out of the tank may also include, for example, a pressure control system or the like. For example, a negative pressure can be created in the discharge device such that the deprotection reagent flows out of the tank.

In a preferred embodiment, during the process of synthesizing the nucleic acid, a deprotecting reagent is added to the sample by the injection device according to the desired oligonucleotide sequence to be synthesized on the one or more solid phase carriers. One or more of the tanks, while the other tanks do not contain a protective reagent.

In a preferred embodiment, the mobile device is a robotic arm.

As used herein, the term "reaction vessel" or "synthetic pool" means any device capable of containing a reagent, including but not limited to tanks, channels, wells, test tubes, cups, dishes, and the like. Preferably, the reaction vessel or synthesis tank is open at one end. The reaction vessel may have any suitable shape, for example, the reaction vessel may be square, spherical, conical, cylindrical, irregular, or the like. The reaction vessel may have any suitable size, for example, its size may be arbitrarily adjusted according to the volume of the reaction solution to be contained, for example, it may be sized to accommodate at least 1 μL, at least 5 μL, at least 10 μL, at least 20 μL, at least 50 μL, at least 100 μL, at least 1 mL, at least 5 mL, at least 10 mL, at least 20 mL, at least 50 mL, at least 100 mL, at least 200 mL, at least 500 mL, at least 1 L or more, or may be sized to accommodate up to 1 μL, up to 5 μL, up to 10 μL, Up to 20 μL, up to 50 μL, up to 100 μL, up to 1 mL, up to 5 mL, up to 10 mL, up to 20 mL, up to 50 mL, up to 100 mL, up to 200 mL, up to 500 mL, up to 1 L or more of reaction solution. The reaction vessel can be made of any suitable material, such as glass, metal such as stainless steel, polymeric materials such as plastic, and the like. It should be understood that the material of the reaction vessel should not adversely affect the reactivity of the reagent.

In the embodiment of the present invention, the mobile device (for example, the robot arm) is controlled by a computer, and the control manner may be an electric control, a magnetic control or an electromagnetic hybrid mode; the moving direction of the mobile device (for example, the mechanical arm) may be horizontal, vertical, Free movement in the direction of the circumference.

An example of typical conditions for solid phase synthesis of oligonucleotides using phosphoramidite monomers or multimers will be described below.

The first step in the preparation of oligonucleotides comprising a solid support represented by the formula (I) deprotection, i.e. removal of a 5'-protecting group represented by R ^1. When R ¹ is an acid labile protecting group, R ^{1 is} removed by treatment of the oligonucleotide with an acid. Preferably the 5'-protecting group is a trityl group such as 4,4'-dimethoxytrityl. When the 5'-protecting group is a trityl group, the oligonucleotide can be removed by treating the oligonucleotide with a solution of dichloroacetic acid, trichloroacetic acid or trifluoroacetic acid in an organic solvent such as dichloromethane, acetonitrile.

The second step of preparing the oligonucleotide comprises coupling the phosphoramidite multimer to the deprotected solid support represented by structural formula II. During the coupling reaction, the -OH or -SH group on the solid support or the 5'-deprotected group and the polymer of the nucleoside or oligonucleotide attached to the solid support are replaced by a -NR ₄ R _{The 5} group reacts. When synthesized in solution, the concentration of the 5'-deprotected nucleotide is typically about 0.02-2 M, and the concentration of the multimer is preferably 5'-deprotected nucleoside or about 5 of the oligonucleotide. -15 equivalents. About 10-20 equivalents of a phosphoramidite activator, such as tetrazole, S-ethylthiotetrazole, dicyanoimidazole or pyridinium salt (such as pyridine chloride), which is equivalent to a 5'-deprotected nucleoside, is often added.鎓) to promote the coupling reaction. The reaction time is usually from about 10 seconds to about 120 seconds.

The third step of preparing the oligonucleotide typically involves capping the unreacted deprotected solid support to render it unreactive in the subsequent coupling step. The sequences that failed in the synthesis were capped such that they were more easily separated from the full length oligonucleotide. Can be any nucleophilic 5'-end group (i.e., -OH, -SH, or -NH ₂₎ and prevent its reaction with the phosphoramidite monomeric or polymeric reaction reagent can be used as capping agent. An acid anhydride such as acetic anhydride or isobutyric anhydride or an acid chloride such as acetyl chloride or isobutyryl chloride is usually used as a blocking agent in the presence of a base.

The fourth step in the preparation of the oligonucleotide comprises oxidizing or vulcanizing the trivalent phosphorus group of the oligonucleotide. In this step, a newly formed nucleus or a 5'-deprotected group of the nucleoside or oligonucleotide attached to the -OH or -SH group or the solid support on the solid support is formed. The trivalent phosphorus bond is oxidized or sulfided.

The oxidation reaction is usually carried out by treating the oligonucleotide with an oxidizing agent, for example, I ₂ in the presence of water, or a peroxide such as t-butyl hydroperoxide in an organic solvent. When I ₂ and water are used, the oxidizing solution typically contains from about 20 to about 100 equivalents of I ₂ in the presence of a base and traces of water. The reaction is carried out in an aprotic polar solvent such as THF, to which is added a base such as a tertiary alkylamine or pyridine and about 1% water. The ratio of aprotic solvent to base is from about 4:1 to about 1:4 (by volume). The oxidation reaction is usually carried out for about 5 seconds to about 2 minutes.

Alternatively, the trivalent phosphorus group can be sulfided with any sulfur transfer reagent known to those skilled in the art for oligonucleotide synthesis. Examples of the sulfur transfer reagent include 3H-benzobisthiazol-3-one 1,1-dioxide (also referred to as "Beaucage reagent"), dibenzoyltetrasulfide, phenylacetyl disulfide, and disulfide. N,N,N',N'-tetraethylthiuram, 3-amino-[1,2,4]-dithiazole-5-thione (see U.S. Patent No. 6,096,881, the disclosure of which is incorporated herein by reference ), or elemental sulfur. Examples of reaction conditions for the sulfidation of oligonucleotides with the above reagents can be found in Beaucage et al., Tetrahedron (1993), 49: 6123, the disclosure of which is incorporated herein by reference in its entirety. 3-Amino-[1,2,4]-dithiazole-5-thione is a preferred sulfur transfer reagent. Typically, the oligonucleotide is made with 3-amino-[1,2,4]-dithiazole-5-thione at a concentration of about 0.04-0.2 M in an organic solvent (eg pyridine/acetonitrile, 1:9 w/w) The solution in contact. The sulfurization reaction is usually carried out for about 30 seconds to about 2 minutes.

Phosphoric acid oligonucleotides can be prepared by oxidizing a trivalent phosphorus group and selecting an internucleotide phosphorus protecting group (for example, a group represented by R ³ ) such that a phosphate group is formed after deprotection of the internucleotide group. . For example, the oxidation of trivalent phosphorus protected by β-cyanoethoxyl followed by alkali deprotection results in the formation of a phosphate group.

The phosphorothioate oligonucleotide can be formed by vulcanizing or oxidizing a trivalent phosphorus group and selecting an internucleotide phosphorus protecting group (for example, a group represented by R ³ ) such that the internucleotide group is deprotected. Prepared by phosphorothioate groups. For example, beta-cyanoethylthio-protected trivalent phosphorus is oxidized and then deprotected with anhydrous basics to form a phosphorothioate group.

The chimeric oligonucleotide may be prepared by oxidizing a trivalent phosphorus group in one or more reaction cycles and vulcanizing the trivalent phosphorus group in one or more additional reaction cycles while appropriately selecting the core. A phospho-phosphorus protecting group (such as the group represented by R ³ ) to form the desired phosphate or phosphorothioate group. Alternatively, chimeric oligonucleotides can be prepared by selecting a polymer in which some internucleotide protecting groups form a phosphorothioate group upon deprotection, such as beta-cyanoethylsulfide. The base protecting group, and other internucleotide phosphorus protecting groups form a phosphate bond upon deprotection, such as a beta-cyanoethoxy protecting group. In this method, the oligonucleotide is oxidized after each coupling step of the reaction cycle.

When it is desired to obtain a 5'-end group in which the protected oligonucleotide is protected, if the capping is carried out, the last step of the reaction cycle may be a capping step; if no capping is performed, the last step of the reaction may be Oxidation or vulcanization step. If desired is a 5'-deprotected oligonucleotide, the reaction cycle can be terminated with a deprotection step. Generally, if the oligonucleotide is to be purified by reverse phase high performance liquid chromatography (HPLC), the 5'-protected oligonucleotide is the desired product. If the oligonucleotide is to be purified by ion exchange chromatography, the 5'-deprotected oligonucleotide is typically the desired product.

In the method of the present invention, the 5'-protected nucleoside or oligonucleotide can be carried on a solid support, usually in an amount of from about 50 to 100 μmol per gram of the carrier. In the coupling step, a solution of a phosphoramidite multimer in an organic solvent (e.g., acetonitrile) at a concentration of usually from about 0.01 to about 1 M, preferably about 0.1 M, is added to the solid support. The carrier to which the 5'-deprotected nucleoside or oligonucleotide is bound is then contacted with the mixture for about 10 seconds to 2 minutes, preferably about 90 seconds.

If the trivalent phosphorus linkage is to be oxidized after completion of the coupling reaction, the solid support and the oxidant, for example with an I ₂ /water mixture or with a peroxide (such as t-butyl hydroperoxide) in an organic solvent (eg THF, Contact in acetonitrile or toluene). A mixture of I ₂ and water is the preferred oxidizing agent. When a mixture of I ₂ and water is used, other water-miscible organic solvents may also be present. Typically, the solid support bound oligonucleotide with the nucleotide conjugate trivalent phosphorus can be contacted with I ₂ in a solvent mixture of water, an aprotic solvent (reagent may be mixed with water) and a base solution. A typical oxidation solution is about 0.01-1.5M, preferably 0.1M of I water / pyridine / tetrahydrofuran solution ₂ (2:20:78) a. The solid support is typically treated with an I ₂ solution for about 5 seconds to 120 seconds, preferably 30 seconds.

Alternatively, the solid support may be contacted with a solution of a sulfur transfer reagent in an organic solvent to vulcanize the trivalent phosphorus group. For example, the solid support can be contacted with a solution of 3H-benzodithiazol-3-one-1,1-dioxide in an organic solvent such as acetonitrile (about 0.05-0.2 M) for about 30 seconds to 2 minutes. .

In the oligonucleotide synthesis of the present invention, the deprotection step is accomplished by contacting the solid support with an acid solution for about 10 seconds to 180 seconds (e.g., 60 seconds). Preferably, the deprotecting agent is selected from the group consisting of a solution of trichloroacetic acid in dichloromethane or a solution of trifluoroacetic acid in acetonitrile. The solid support can optionally be contacted with the phosphoramidite activator solution for about 10 seconds to 120 seconds. This reaction cycle can be repeated one or more times until the desired length of oligonucleotide is synthesized. When the reaction cycle ends with a capping step or an oxidation or sulfurization step, a 5'-protected oligonucleotide is obtained. When the reaction cycle is terminated with a deprotection step, a 5'-deprotected oligonucleotide is obtained.

In an embodiment of the invention, the method further comprises the step of separating the synthetic oligonucleotide from the solid support. Preferably, the oligonucleotide can be removed from the solid support using an aminolysis process. The reagent for the aminolysis method may be selected from any one of ammonia water, ammonia gas, and methylamine; the aminolysis temperature may be 25, 60, 90 ° C or any temperature therebetween; the aminolysis time is usually from about 0.5 hour to about 18 Hours or longer, such as 2h, 5h, 10h, 18h or 24h. Subsequently, the method can further comprise purifying the synthetic oligonucleotide using a purification method selected from the group consisting of desalting, MOP, PAGE, PAGE Plus or HPLC.

Advantageous technical effects of the present invention:

The nucleic acid synthesis method proposed by the invention realizes low-cost, high-efficiency and accurate double-base nucleic acid synthesis by using the "synthesis pool" method for the first time. The nucleic acid synthesis method of the invention can recycle the reaction reagent in the "synthesis cell" by precisely controlling the "synthesis needle" group moving, immersing and lifting, without greatly designing complicated reagent input and output pipelines, thereby greatly reducing Material cost. In addition, the method can ensure the flux and yield of nucleic acid synthesis by designing different "synthetic needle" groups. By precisely controlling the "synthetic needle" group, the synthesis time can be precisely controlled and shortened, and the chemical synthesis efficiency can be fully utilized. The design of the pool ensures no cross-contamination and thus controls low error rates. Further, compared with the conventional single-base nucleic acid synthesis, under the same nucleic acid synthesis cycle number, the original four-step cycle method is used for 100 cycles to obtain 100 bp, based on the "synthesis pool" double-base nucleic acid synthesis strategy, Under the premise of ensuring low cost and low error rate, it can now produce a 200 bp nucleic acid single chain, breaking the technical bottleneck faced by the single base nucleic acid synthesis technology widely used in commercial services.

The novel double-base nucleic acid synthesis design scheme based on "synthesis pool" proposed by the invention firstly realizes the method of synthesizing double base nucleic acid by controlling the "synthetic needle" by a pre-design program, and verifies the practicability of the method; In this nucleic acid synthesis, porous glass is used as a solid phase carrier, which ensures low error rate and high reaction efficiency; more importantly, the synthesis reagent can be reused through the "synthesis cell", which greatly reduces the amount of reagent used, and at the same time There is no need to lay complex reagent input and output lines separately, which greatly reduces the synthesis cost. In addition, the “synthetic needle” can be expanded on a large scale and easily integrated with other devices due to its small structure and simple fabrication, such as polymerase chain. The reaction device makes the nucleic acid synthesis more convenient and efficient; finally, the "synthesis pool" double base nucleic acid synthesis method can also be extended to the "synthesis pool" of the three-base nucleic acid synthesis, the "synthesis pool" of the four-base nucleic acid Synthesis, etc. A schematic of the "synthetic pool" nucleic acid synthesis system is shown in Figure 2.

In summary, the nucleic acid synthesis method of the present invention improves the reaction efficiency and ensures a low error rate on the basis of greatly reducing the cost of nucleic acid synthesis. At the same time, the "synthetic needle" group in the invention has the characteristics of being expandable and easy to integrate, and can improve the synthesis flux through optimization design, and can also be combined with module automation for function expansion, such as assembling downstream polymerase chain reaction and gene assembly technology flow. . The nucleic acid method of the invention integrates the advantages of the existing synthetic methods, skillfully avoids the problems exposed by other methods, and lays a foundation for the future development of the third generation low-medium-high-flux synthesizer.

DRAWINGS

Figure 1 shows an example of four single base monomers and 20 double base monomers. Wherein Bz is a benzoyl group and ib is an isobutyryl group.

Figure 2 shows a schematic of a "synthetic pool" nucleic acid synthesis system. The blocks shown in the left three columns are synthetic pools, and the area shown in the fourth column is the robotic arm control station, which controls the movement, immersion and lifting of the "synthetic needle" group by controlling the robot arm.

Figures 3a-d show an exemplary embodiment of a "synthetic pool" synthetic oligonucleotide.

Figure 4 shows the glass flakes after completion of the synthesis of the T5 primer in Example 1.

Figure 5 shows a schematic diagram of the aminolysis in Example 1.

Figure 6a shows the HPLC profile of the T5 reference in Example 1; Figure 6b shows the HPLC profile of the T5 primer in Example 1.

Fig. 7 shows a glass piece after completion of the synthesis of the T10 primer in Example 2.

Figure 8 shows a schematic diagram of the aminolysis in Example 2.

Figure 9a shows the HPLC profile of the T10 reference in Example 2; Figure 9b shows the HPLC profile of the T10 primer in Example 2.

Detailed ways

Example 1

The specific steps of synthesizing oligonucleotides by the synthetic pool method are described below by taking single base synthesis as an example.

The glass sheet modification process is as follows:

Take a piece of glass (Shitai, pathological microscope slide, 25*75mm) and wash it twice in acetone. After drying, do PLASMA in the plasma cleaner (the chamber gas is oxygen, the vacuum is 700-800mtorr, The time is 10 min), CVD is carried out within 5 min after the completion of PLASMA (vacuum degree is -0.8 kg/cm2, raw material 200 uL APTMS, time is 30 min), after vapor deposition is completed, it is washed once with deionized water, acetone is washed once, and blown dry. The above-mentioned glass piece to be dried was placed in a solution of 750 mg of Linker, 400 mg of HATU, 800 μL of DIPEA dissolved in 50 mL of acetonitrile, and stirred overnight in a vertical stirrer. The glass piece was taken out, washed three times with acetonitrile, and washed three times with acetone. Dry, the glass piece is finished. The Linker graft density of the modified glass sheet was measured by an ultraviolet spectrophotometer to be 0.003636 nmol/mm ² .

The oligonucleotide synthesis process is as follows:

One oligonucleotide sequence to be synthesized is shown below, and the oligonucleotide was synthesized on one modified glass slide.

Sequence to be synthesized: TTTTT, designated as T5 primer.

Each piece of modified glass piece is a solid phase carrier according to the present invention, and only the first deprotection can be carried out to carry out the subsequent coupling step, and if the chain length of the nucleic acid sequence to be synthesized is short (cycle number ≤ 25), the cap in the experimental operation The steps can be omitted. If a longer chain length nucleic acid sequence is to be synthesized (cycle number > 25), a capping step is required to reduce the error rate to obtain a sufficient number of target nucleic acids.

1) Cut a piece of glass about 1.0cm*2.5cm into the TCA for 60s to protect it and take it out;

2) Washing in acetonitrile (No. 1) for 60 s, taking out, and then washing in acetonitrile (No. 2) for 60 s, and taking out;

3) immersed in a solution of 0.1 M phosphoramidite monomer T and an activator in a volume ratio of 1:1 for 90 s, and taken out;

4) immersed in an oxidizing agent for 30 seconds for oxidation, and taken out;

5) Washing in acetonitrile (No. 3) for 60 s, taking out and then washing in acetonitrile (No. 4) for 60 s;

6) At this point, one cycle is completed. After repeating the above process 5 times, the glass piece is immersed in the TCA for 60 seconds to be protected and taken out;

7) Washing in acetonitrile (No. 1) for 60 s, taking out and then washing in acetonitrile (No. 2) for 60 s, and finally obtaining DMT OFF T5 primer;

The specific procedure, reagents used and immersion time are shown in the table below:

8) Cut the synthesized glass piece into small pieces, transfer it to a small glass bottle containing 700 μL of ammonia water, screw the lid, place it in a 55 ° C water bath, dissolve the ammonia solution, remove the ammonia water, and add 100 μL. Ionized water, sonicated, centrifuged, and the supernatant was measured using a nanodrop instrument;

9) The supernatant purity was analyzed using HPLC, and the results of the T5 reference and the T5 primer are shown in Figures 6A and 6B, respectively.

By comparing the HPLC spectra of the T5 reference product and the T5 primer synthesized by the soaking method of the present invention, it was found that there was a distinct T5 product peak at around 34.48 min, indicating that the single-base synthesis strategy by soaking method can successfully synthesize the length of T5. Oligonucleotides, and derived from the peak areas shown, have a purity of 52.1% of the synthesized T5 product. The amount of T5 primer synthesis was 0.696 nmol.

Example 2

Hereinafter, a specific step of synthesizing an oligonucleotide by a synthetic pool method will be described by taking a two-base DNA synthesis as an example.

The glass sheet modification process is as follows:

Take a slide (Shitai, pathological microscope slide, 25*75mm) and wash it twice in acetone. After drying, do PLASMA in the plasma cleaner (the chamber gas is oxygen, the vacuum is 700-800mtorr). , time is 10min), CVD is carried out within 5min after the completion of PLASMA (vacuum degree is -0.8kg/cm2, raw material 200μL APTMS, time is 30min), after vapor deposition is completed, it is washed once with deionized water, acetone is washed once, Blow dry. The above-mentioned glass piece to be dried was placed in a solution of 750 mg of Linker, 400 mg of HATU, 800 μL of DIPEA dissolved in 50 mL of acetonitrile, and stirred overnight in a vertical stirrer. The glass piece was taken out, washed three times with acetonitrile, and washed three times with acetone. Dry, the glass piece is finished. The Linker graft density of the modified glass sheet was measured by an ultraviolet spectrophotometer to be 0.003636 nmol/mm ² .

The oligonucleotide synthesis process is as follows:

Sequence to be synthesized: TTTTTTTTTT, named T10 primer.

The double base synthetic monomer used is: DMT-dT-dT.

3) immersed in a solution of 0.1 M phosphoramidite monomer DMT-dT-dT and an activator in a volume ratio of 1:1 for 90 s, and taken out;

4) immersed in an oxidizing agent for 30 seconds for oxidation, and taken out;

7) Wash in acetonitrile (No. 1) for 60 s, take out and wash in acetonitrile (No. 2) for 60 s, and finally obtain DMT OFF T10 primer.

9) The supernatant purity was analyzed using HPLC, and the results of the T10 reference and the T10 primer are shown in Figures 9A and 9B, respectively.

By comparing the HPLC spectra of the T10 reference product and the T10 primer synthesized by the soaking method of the present invention, it was found that there was a distinct T10 product peak at around 41.86 min, indicating that the length of T10 can be successfully synthesized by the double base synthesis strategy of the soaking method. Oligonucleotides, and from the peak areas shown, the synthesized T10 product was 51.8% pure. The amount of T10 primer synthesis was 0.308 nmol.

Claims

A method of synthesizing an oligonucleotide, the method comprising:

a) providing a solid phase carrier represented by formula (I) or a stereoisomer of formula (I),

among them:

Nu is a single bond or

Wherein X 2 in Nu is attached to R 13 ;

X 1 is independently -O- or -S-;

X 2 is independently -O-, -S- or -NR-;

X 3 is independently -O-, -S-, -CH 2 - or -(CH 2 ) 2 -;

X 4 is independently =O or =S;

R 1 is a protecting group;

R 2 is independently -H, -F, -NHR 6 , -CH 2 R 6 or -OR 6 ;

R 3 is independently -OCH 2 CH 2 CN, -SCH 2 CH 2 CN, a substituted or unsubstituted aliphatic group, -OR 7 or -SR 7 ;

R is -H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or an amine protecting group;

R 6 is -H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, or a protecting group;

R 7 is a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group;

Each B is independently a base that is modified or unmodified;

R 13 is a solid phase carrier or -Y 1 -LY 1 -R 14 ;

Y 1 is a single bond, a double bond, -C(O)-, -C(O)NR 17 , -C(O)O-, -NR 17 - or -O-;

L is a single bond, a double bond, a substituted or unsubstituted aliphatic group, or a substituted or unsubstituted aryl group;

R 17 is -H, a substituted or unsubstituted aliphatic group, or a substituted or unsubstituted aryl group;

R 14 is a solid phase carrier; and

q is 0 or a positive integer;

b) contacting the solid support with a deprotecting agent in a first reaction vessel containing a deprotecting agent to form a deprotected solid support represented by formula (II) or a stereoisomer of formula (II) ,

Wherein X 5 is -OH or -SH; the other groups are as defined above;

c) subjecting the solid support to a phosphoramidite activator and a sub-reagent in a second reaction vessel comprising a phosphoramidite activator and a phosphoramidite monomer or multimer or a stereoisomer thereof of formula (III) Phosphoramide monomer or multimer or a stereoisomer thereof,

among them:

R 4 and R 5 are each independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl group;

R 4 and R 5 together with the nitrogen to which they are bonded form a heterocycloalkyl or heteroaryl group, wherein the heterocycloalkyl or heteroaryl group is preferably a five or six membered ring;

n is 0 or a positive integer;

Other groups are as defined above;

Wherein the phosphoramidite monomer or polymer represented by the formula (III) or a stereoisomer thereof is coupled with the deprotected solid phase carrier or stereoisomer represented by the formula (II), thereby forming a formula ( a first intermediate or a stereoisomer thereof as shown in IV),

e) contacting the solid support with an oxidizing agent or a vulcanizing agent in a fourth reaction vessel containing an oxidizing agent or a vulcanizing agent, wherein the trivalent phosphorus group in the first intermediate is oxidized or vulcanized to form the first formula (V) a second intermediate or a stereoisomer thereof;

Where r is a positive integer;

Wherein, the first reaction vessel, the second reaction vessel and the fourth reaction vessel are independent of each other.
The method of claim 1 wherein between steps c) and e), further comprising step d):

d) contacting the solid support with a blocking agent in a third reaction vessel comprising a blocking agent, wherein the step (c) of reacting with the phosphoramidite monomer or polymer in step c) is carried out The X 5 group of the protected solid support or stereoisomer is capped,

The third reaction vessel and the first reaction vessel, the second reaction vessel, and the fourth reaction vessel are independent of each other.
The method of claim 1 or 2, wherein said method further comprises step f):

f) repeating steps b), c), e) or b) to e) one or more times, wherein the final step is step b), d) or e), whereby the desired oligonucleotide is synthesized.
The method of claim 1 wherein said solid support is two or more solid supports of the same or different.
The method of claim 4, wherein step b) comprises including one or more of said two or more solid phase carriers according to the desired oligonucleotide sequence to be synthesized on each solid phase support The first reaction vessel of the deprotecting agent is contacted with a deprotecting agent.
The method of claim 2 further comprising a washing step between steps b and c and between steps d and e.
The method of claim 6 wherein said washing step washes the solid support by contacting said solid support with a wash reagent in a fifth reaction vessel comprising a detergent,

The fifth reaction vessel is independent of the first reaction vessel, the second reaction vessel, the third reaction vessel, and the fourth reaction vessel.
The method of claim 3, wherein in step f), the deprotecting agent, said second reaction in said first reaction vessel is repeated each time steps b), c), e) or b) to e) are repeated The activator in the container is repeated with the phosphoramidite monomer or multimer or a stereoisomer thereof, the capping agent in the third reaction vessel, and/or the oxidizing agent or vulcanizing agent in the fourth reaction vessel use.
The method of any one of claims 1-8, wherein the method further comprises the step of separating the synthetic oligonucleotide from the solid support.
The method of any one of claims 1-9, wherein n = 1, 2 or 3.
The method of any one of claims 1-9, wherein n=1.
The method of any one of claims 1-9, wherein X 1 is -O-; said X 2 is -O-; said X 3 is -O-; said X 4 is =0; And/or the X 5 is -OH.
Process according to any one of claims 1-9, characterized in that the phosphoramidite activator is selected from the group consisting of tetrazole, S-ethylthiotetrazole, dicyanoimidazole or pyridinium salt.
The method of any of claims 1-9, wherein the oxidizing agent is selected from the group consisting of iodine solutions.
Process according to any one of claims 1-9, characterized in that the vulcanizing agent is selected from the group consisting of 3-amino-[1,2,4]-dithiazole-5-thione or 3H-benzodithiazole-3 - Ketone 1,1-dioxide.
Process according to any one of claims 1-9, characterized in that the deprotecting agent is selected from a solution of trichloroacetic acid in dichloromethane or a solution of trifluoroacetic acid in acetonitrile.
Process according to any one of claims 1-9, characterized in that said R 1 is selected from substituted or unsubstituted trityl, monoalkoxytrityl, dialkoxytrityl, tri Alkoxytrityl, tetrahydropyranyl or 9-phenylxanthene.
Process according to any one of claims 1-9, characterized in that said R 2 is selected from -H, -O or -OCH 2 CH 2 OMe.
Process according to any one of claims 1-9, characterized in that said R 3 is -OCH 2 CH 2 CN or -SCH 2 CH 2 CN.
Process according to any one of claims 1-9, characterized in that said R 4 and R 5 are each isopropyl.
Process according to any one of claims 1-9, characterized in that said R 7 is o-chlorophenyl or p-chlorophenyl.
The method of claims 1-9, wherein said method further comprises a treatment with a base synthetic oligonucleotides to remove from -OCH 2 CH 2 CN or -SCH 2 CH 2 CN -CH 2 CH 2 CN in .
The method according to any one of claims 1 to 9, wherein the phosphoramidite monomer or polymer represented by formula (III) or a stereoisomer thereof is a phosphoramidite compound represented by formula (VI) Body or multimer or a stereoisomer thereof,

Wherein B and R 2 have the same definitions as in formula I;

R 8 is a substituted or unsubstituted trityl group;

R 10 and R 11 are each independently a substituted or unsubstituted aliphatic group;

m is 0, 1, or 2.
The method of claim 23 wherein said R 8 is 4,4'-dimethoxytrityl.
The method of claim 23 wherein said R 10 and R 11 are isopropyl.
The method of claim 23, wherein the phosphoramidite monomer or polymer represented by formula (III) or a stereoisomer thereof is selected from one of the following 20 compounds or a stereoisomer thereof:

Wherein Bz is a benzoyl group and ib is an isobutyryl group.
The method of any one of claims 1-9, wherein the synthesized oligonucleotide has a length greater than or equal to 100 nt.
The method of any one of claims 1-9, wherein the synthesized oligonucleotide has a length greater than or equal to 150 nt.
The method of any one of claims 1-9, wherein the synthesized oligonucleotide has a length greater than or equal to 200 nt.
A system for nucleic acid synthesis comprising:

One or more solid phase carriers as shown in formula (I) or stereoisomers represented by formula (I),

One or more reaction vessels containing a deprotecting agent,

One or more reaction vessels comprising a phosphoramidite activator and a phosphoramidite monomer or multimer represented by formula (III) or a stereoisomer thereof,

One or more reaction vessels containing an oxidizing agent or a vulcanizing agent,

a mobile device for moving the one or more solid phase carriers between reaction vessels, and

A control system for controlling movement of the mobile device.
The system of claim 30 wherein said system further comprises one or more reaction vessels comprising a blocking agent.
The system of claim 30 wherein said system further comprises one or more reaction vessels comprising a detergent.
The system of claim 30 wherein said one or more solid phase carriers are detachably secured to said mobile device.
The system of claim 30 wherein said mobile device controls said one or more solid phase carriers to move simultaneously between the respective reaction vessels.
The system of claim 30 wherein said mobile device is a robotic arm.
The system of claim 30 wherein said control system is a computer operated programmatic control system.
A system for nucleic acid synthesis comprising:

One or more solid phase carriers as shown in formula (I) or stereoisomers represented by formula (I),

a reaction vessel for receiving a deprotection reagent having one or more separate tanks, and the number of said tanks being at least equal to the number of said solid phase supports,

One or more reaction vessels comprising a phosphoramidite activator and a phosphoramidite monomer or multimer represented by formula (III) or a stereoisomer thereof,

One or more reaction vessels containing an oxidizing agent or a vulcanizing agent,

a mobile device for moving the one or more solid phase carriers between reaction vessels, and

A control system for controlling movement of the mobile device.
The system of claim 37 wherein said one or more separate troughs each correspond to a solid support.
The system of claim 37 or 38, characterized by one or more of the following:

a) the system further comprises an injection device for adding a deprotection reagent to the tank,

b) the injection device is an injector containing a deprotection reagent,

c) the injection device is a reaction vessel containing a deprotection reagent in fluid communication with the tank,

d) the injection device further comprises means for controlling the flow of the deprotection reagent into the tank,

e) the means for controlling the flow of deprotection reagent into the tank comprises a valve,

f) the means for controlling the flow of deprotection reagent into the tank comprises a pressure control system,

g) the system further comprising a discharge device for discharging the deprotection reagent from the tank,

h) the discharge device is a pipette,

i) the discharge device is a fluid passage in fluid communication with the tank,

j) the discharge device further comprises means for controlling the flow of the deprotection reagent out of the tank,

k) the means for controlling the deprotection reagent to flow out of the tank comprises a valve,

l) the means for controlling the deprotection reagent to flow out of the tank comprises a pressure control system,

m) the system further comprises one or more reaction vessels containing detergent,

n) adding, during the process of synthesizing the nucleic acid, one or more deprotecting reagents to the tank by the injection device according to the desired oligonucleotide sequence to be synthesized on the one or more solid phase carriers In the other tanks, the other tanks do not contain protective reagents.

o) the mobile device controls the one or more solid phase carriers to move simultaneously between the respective reaction vessels,

p) the one or more solid phase carriers are detachably fixed to the mobile device,

q) the mobile device is a robotic arm,

r) the control system is a computerized programmatic control system,

s) The system further comprises one or more reaction vessels comprising a blocking agent.