CN117701658A - Method for improving purity and yield of oligonucleotide chain enzymatic synthesis - Google Patents

Abstract

The invention discloses a method for improving the enzymatic synthesis purity and yield of an oligonucleotide chain, which comprises (1) adopting a bioenzyme-based method to realize the base extension of the oligonucleotide chain by taking dNTP-3' -R as a raw material on a solid phase carrier; (2) Coupling ddNTPs with TdT at the 3' -end of the oligonucleotide strand where no base extension occurs to effect a capping and blocking reaction; (3) Deprotection is then performed to deprotect the-R at the 3' end of the base extended oligonucleotide strand to form activated-OH; (4) Repeating steps (1) to (3) until synthesis of the desired oligonucleotide strand is achieved. The invention can realize the efficient blocking reaction of hydroxyl groups which do not undergo extension reaction in each round of oligonucleotide chain enzymatic synthesis process, thereby improving the purity and yield of the enzymatic synthesis end product.

Description

A method for improving the purity and yield of enzymatic synthesis of oligonucleotide chains

Technical field

The invention belongs to the technical field of DNA synthesis, and specifically relates to a method for improving the purity and yield of enzymatic synthesis of oligonucleotide chains.

Background technique

DNA is the main carrier of life information, and RNA is the intermediate carrier of genetic information transmission. They play a vital role in the life process. Since the last century, humans have had an increasingly mature and comprehensive understanding of DNA and RNA, including the composition, structure, properties, and functions of DNA. This also laid a solid foundation for scientific researchers to use DNA and RNA as "sharp tools" for studying life sciences.

Oligonucleotides generally refer to short strands of DNA or short strands of RNA synthesized and manufactured under laboratory conditions. Over the past nearly 70 years, de novo oligonucleotide synthesis technology has been widely used in most biological research, significantly improving humankind's ability to understand and transform life.

To this day, the phosphoramidite solid-phase oligonucleotide synthesis method proposed in the 1980s (referred to as the phosphoramidite method) still occupies a dominant position. The phosphoramidite method uses dimethoxytrityl (DMT) as the protective group of phosphoramidite monomers to connect adjacent nucleotides through the synthesis of 5' → 3' phosphodiester bonds, which usually includes the following Four steps:

(1) Deprotection: Use trichloroacetic acid to remove the 5’-DMT protecting group of phosphoramidite on the solid support to obtain the free 5’-hydroxyl group;

(2) Coupling: A new round of 5-DMT protected phosphoramidite monomer is introduced, and its 3' end is converted to an intermediate under the action of the activator tetrazole, which can be coupled with the free 5'-hydroxyl group to form Phosphite triesters;

(3) Capping: During the coupling step, there will be a small amount of unreacted 5'-hydroxyl groups (<2%). Acetic anhydride and N-methylimidazole can be added to acetylate the unreacted 5'-hydroxyl groups. Terminate subsequent reactions, leaving shorter fragments of oligonucleotide chains that can be separated during purification;

(4) Oxidation: Use iodine to oxidize phosphite triester to generate a more stable phosphate triester, producing a cyanoethyl protected phosphate skeleton; then add the target nucleoside phosphoramidite monomers sequentially from 5'→3' , cycle the above four synthesis steps, and finally use concentrated ammonia to remove all protective groups on the complete product (including DMT on the monomer and cyanoethyl on the skeleton) at one time, and remove, desalt, and After purification, the oligonucleotide chain can be obtained;

However, after decades of technical optimization and performance improvement, this traditional chemical synthesis method has always had the bottleneck problem of insufficient synthesis length (the upper limit is about 200nt). At the same time, the coupling efficiency is low, the error rate is high, and the cost is high. High, toxic and harmful synthetic reagents, poor biocompatibility and other shortcomings also limit its further application. The technical difficulties of chemical synthesis have also prompted scientific researchers to turn to the technical route of biological enzymatic synthesis in nature.

Compared with chemical synthesis, enzymatic synthesis has several potential advantages (NAT. METHODS, 2014, 11(5): 499-507): (1) Enzymatic synthesis has a synthesis length that far exceeds that of phosphoramidite synthesis technology, Speed and efficiency, it is expected that the synthesis of oligonucleotides of at least 400nt length can be achieved, the efficiency can reach 99.5%, and the error rate is less than 0.5%; (2) The enzyme can achieve efficient enzymatic synthesis under mild action, compared with chemical synthesis Using a variety of toxic and harmful organic reagents, enzymatic synthesis can not only reduce damage to oligonucleotides and the production of by-products, adapt to more applications that require high biocompatibility, but also greatly reduce hazardous waste. The production is environmentally friendly; (3) The enzymatic synthesis method has simple steps and will greatly reduce the time and cost of single base synthesis. At present, de novo synthesis of template-free oligonucleotides based on terminal deoxynucleotidyl transferase has been reported (NAT. BIOTECH., 2018, 36: 645-650; ACS CATAL., 2022, 12(5) ): 2988-2997), all adopt a two-step method to achieve enzymatic DNA single base extension, namely "extension-deprotection".

Although the two-step method makes enzymatic synthesis simple and efficient, the lack of a capping step means that the majority of errors generated in the enzymatic synthesis process are deletions (about 3-10%) (NAT. BIOTECH., 2018, 36: 645-650; ACS CATAL., 2022, 12(5): 2988-2997). This has greatly affected the purity and yield of the final product of enzymatic DNA synthesis, and weakened the advantages of high efficiency, high fidelity, long length, and high efficiency of enzymatic synthesis in oligonucleotide synthesis.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a method for improving the purity and yield of enzymatic synthesis of oligonucleotide chains.

The definitions of key technical words in the present invention are as follows:

DNA (Deoxyribonucleic acid): deoxyribonucleic acid;

RNA (Ribonucleic acid): ribonucleic acid;

dNTP (Deoxynucleotide triphosphates): deoxyribonucleoside triphosphates;

ddNTP (Dideoxynucleotide triphosphates): 2′, 3′-dideoxynucleoside triphosphates, the hydroxyl group (-OH) at the 3' position of deoxyribonucleoside triphosphates is replaced by a hydrogen group (-H), and subsequent coupling cannot be extended;

dNTP-3'-R: The hydroxyl group (-OH) at the 3' position of the deoxyribonucleoside triphosphate is replaced by the protecting group R to prevent uncontrolled dNTP extension, a typical protection in enzymatic DNA synthesis reactions. The groups are aminooxy (-OHN ₂ ), oxyazidomethyl (-OCN ₃ ) and 2-aminoethyl-3-propionyl (-Aep);

TdT (Terminal deoxynucleotidyl transferase): terminal deoxynucleotidyl transferase, a template-independent DNA polymerase that catalyzes the binding of dNTP or ddNTP to the 3’ hydroxyl end of DNA molecules;

E-ZaTdT (Engineered Zonotrichia albicollis TdT): Engineered ZaTdT enzyme. The wild-type TdT catalytic cavity cannot adapt to the reversible terminating nucleotide. Through enzyme engineering methods, a ZaTdT with catalytic activity much higher than ordinary TdT was screened. And reshape its catalytic cavity to better adapt to the reversible termination extension of 3'-ONH ₂ -modified nucleotides, achieving de novo synthesis of enzymatic oligonucleotide chains.

The technical solution of the present invention is as follows: a method for improving the purity and yield of enzymatic synthesis of oligonucleotide chains, including the following steps:

(1) Use a bioenzymatic method to achieve base extension of the oligonucleotide chain on a solid-phase carrier using dNTP-3’-R as the raw material;

(2) Use TdT to couple ddNTP with -OH at the 3’ end of the oligonucleotide chain that has not undergone base extension to achieve a capping reaction;

(3) Then perform deprotection to deprotect the -R at the 3' end of the oligonucleotide chain where base extension occurs to form activated -OH;

(4) Repeat steps (1) to (3) until the synthesis of the desired oligonucleotide chain is achieved.

The principle of the above capping and blocking reaction is: TdT is a kind of catalytic addition of dNTP or ddNTP to the 3'-OH end of the oligonucleotide chain without a template. Compared with dNTP, due to the smaller spatial position of ddNTP, TdT has a higher catalytic coupling efficiency at the hydroxyl end, so it can react more efficiently with the hydroxyl groups that did not react in the extension step. At the same time, because its 3' end is hydrogen (-H), it can react more efficiently with the hydroxyl group in the next round. No further TdT catalytic coupling can occur in the extension reaction, achieving the blocking effect of the capping reaction. Subsequent purification steps can be used to separate and remove the oligonucleotide chain that has not reached the target fragment length, so that the oligonucleotide chain can be enzymatically synthesized. The product has higher purity and yield.

In a preferred embodiment of the present invention, the ddNTP includes ddATP, ddTTP, ddCTP, ddGTP and ddUTP.

In a preferred embodiment of the present invention, the dNTP-3'-R includes dNTP-3'-ONH ₂ (oxyamino), dNTP-3'-OCN ₃ (oxyazidomethyl) and dNTP- 3'-Aep (2-aminoethyl-3-propionyl).

In a preferred embodiment of the present invention, the TdT in step (2) is selected from wild-type TdT or E-ZaTdT.

In a preferred embodiment of the invention, the oligonucleotide chain is single-stranded DNA, double-stranded DNA or single-stranded RNA.

Further preferably, the oligonucleotide chain is single-stranded DNA.

More preferably, the base extension in step (1) is DNA single base extension based on E-ZaTdT. The principle is: E-ZaTdT, which has been screened and modified through enzyme engineering methods, has a reversible termination extension that can catalyze the coupling of 3'-modified aminooxy (-ONH ₂ ) nucleotides. E-ZaTdT and E-ZaTdT are added to the end of single-stranded DNA. Specific 3'-ONH ₂ modified nucleotides can perform a single base extension reaction at the end of the DNA chain in the presence of divalent cations (cobalt ions Co ²⁺ ).

In a preferred embodiment of the present invention, the deprotection in step (2) is performed in an acidic sodium nitrite solution. The principle is: since the purpose of deprotection is to react the 3' end with the aminooxy (-ONH ₂ ) protecting group to generate a hydroxyl group so that it can be extended in the next round of reaction, it can be submerged under acidic conditions. Sodium nitrate exhibits oxidizing properties and reacts with aminooxy groups to generate hydroxyl groups to achieve deprotection reaction.

Further preferably, the sodium nitrite solution contains non-ionic surfactant.

More preferably, the nonionic surfactant is Triton X-100 or Tween.

The beneficial effects of the present invention are:

1. The present invention can realize efficient blocking reaction of hydroxyl groups that have not undergone extension reaction in each round during the enzymatic synthesis of oligonucleotide chains, thereby improving the purity and yield of the final product of the enzymatic synthesis.

2. The present invention uses TdT to perform the capping reaction, and still uses a biological enzyme reaction system, which does not affect the original enzymatic synthesis system and is suitable for the enzymatic synthesis reaction system of oligonucleotide chains.

3. The present invention uses TdT coupled ddNTP for capping reaction. The reaction efficiency is higher than that of TdT coupled or extended dNTP. The required substrate concentration is low, the required reaction time is short, and it has the advantages of efficient blocking.

4. Compared with the traditional chemical capping reaction, the capping reaction based on TdT coupled ddNTP of the present invention can not only be efficiently adapted to the enzymatic synthesis system, but also fully compatible with the existing enzymatic synthesis system. All reagents are non-toxic and harmless. , has the advantages of low consumption and low pollution.

Description of the drawings

Figure 1 is a reaction principle diagram of Example 1 of the present invention.

Figure 2 is a diagram of experimental results of Example 1 of the present invention.

Detailed ways

The technical solution of the present invention will be further illustrated and described below through specific embodiments and in conjunction with the accompanying drawings.

Example 1

As shown in Figure 1, this embodiment performs oligonucleotide chain synthesis, which specifically includes the following steps:

(1) Streptavidin magnetic beads coupled with biotin-priming chain:

In this example, streptavidin magnetic beads are used as solid-phase carriers based on E-ZaTdT to achieve single-base bioenzymatic synthesis of DNA, which specifically includes:

a. Streptavidin magnetic bead cleaning: In a centrifuge tube, take 20 μL of streptavidin (SA) modified magnetic beads with a concentration of 10 mg/mL, mix with 180 μL of magnetic bead cleaning and binding buffer, and mix Place the centrifuge tube on a magnetic stand and let it stand for 3 minutes. Discard the supernatant. Repeat this step three times.

b. Initiator chain coupling: In the above-mentioned centrifuge tube, add 197 μL of magnetic bead cleaning binding buffer and resuspend, and then add 3 μL of Biotin-Initiator biotin-initiator chain solution with a concentration of 100 μM (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.), perform a binding reaction between biotin and streptavidin, place it on a rotating incubator, and incubate for 30 minutes.

c. Cleaning of priming chain magnetic beads: Wash the incubated magnetic beads three times with 200 μL of ultrapure water. After the cleaning is completed, add 20 μL of ultrapure water and resuspend.

At this point, the solid phase carrier preparation for oligonucleotide chain synthesis is completed.

(2) Oligonucleotide chain synthesis - extension a. Prepare extension master mix: Take 1 centrifuge tube and add 0.5mg/mL E-ZaTdT (see Enzymatic DNA Synthesis by Engineering Terminal DeoxynucleotidylTransferase, ACS Catal. 2022, 12, 5, 2988-2997), 0.25mM 3'-ONH ₂ -dNTPs (the bases required for extension in this round), 0.25mM CoCl ₂ , 100mM NaCl, 0.01% v/v Triton X-100 and 50mM PBS buffer ( pH6.8);

b. Pre-incubation: Place the centrifuge tube at 40°C and pre-incubate for 5 minutes;

c. Extension: Take 20 μg of initiator magnetic beads, add 10 μL of the above extension premix, place it in a constant temperature shaker, set the temperature to 40°C, and the rotation speed to 1400 rpm, react for 10 minutes, and complete the extension.

(3) Oligonucleotide chain synthesis-capping

a. Prepare capping premix: Take a centrifuge tube and add 1U/μL TdT, 0.1mM ddNTP (N can be any base such as A, T, C, G, etc.), 0.25mM CoCl ₂ , 100mM NaCl, 0.01% v/v Triton X-100 and 50mM PBS buffer (pH6.8);

b. Pre-incubation: Place the centrifuge tube at 40°C and pre-incubate for 5 minutes;

c. Capping: After magnetic separation of the above-mentioned extended magnetic beads to remove the supernatant, add 10 μL of the above-mentioned capping premix, place it in a constant temperature shaker, set the temperature to 40°C, and set the rotation speed to 1400rpm. The reaction is completed for 1 minute. extend.

(4) Oligonucleotide chain synthesis - deprotection

a. Prepare deprotection preparatory solution: Take a centrifuge tube, add 0.7M sodium nitrite, and use nitrous acid (prepared by adding acetic acid to sodium nitrite) to adjust the pH of the solution to 5.0, and add 0.01% Triton X-100, keep in ice for later use;

b. Deprotection: After the above-mentioned capped magnetic beads are magnetically separated to remove the supernatant, add 10 μL of the above-mentioned deprotection pre-prepared solution, shake and incubate for 1 minute to complete deprotection;

(5) Oligonucleotide chain synthesis and cleaning one by one

a. Prepare the cleaning premix: Take a centrifuge tube and add 0.01% v/v Triton X-100 and 50mM PBS buffer (pH 6.8);

b. Washing: Perform magnetic separation on the above-mentioned deprotected magnetic beads to remove the supernatant, add 10 μL of the above-mentioned cleaning pre-preparation solution, shake and incubate for 0.5 min, repeat 2 times.

At this point, a single round of synthesis of the oligonucleotide chain is completed. Repeat the above steps (1) to (5) according to the specified oligonucleotide chain sequence to complete the synthesis of the oligonucleotide chain.

The specific effects of this embodiment are shown in Figure 2. Figure 2 shows the impact of capping and non-capping on the stepwise yield between 4 rounds, 10 rounds and 18 rounds of enzymatic synthesis of DNA. As can be seen from the figure, The synthesis method of this embodiment, especially the capping in step (3), can significantly improve the stepwise yield of DNA enzymatic synthesis, especially when the synthetic chain length increases.

The above are only preferred embodiments of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made based on the patent scope of the present invention and the content of the specification should still be covered by the present invention. In the range.

Claims (10)

1. A method for improving the purity and yield of enzymatic synthesis of oligonucleotide chains, which is characterized in that it includes the following steps: (1) Use a bioenzymatic method to achieve base extension of the oligonucleotide chain on a solid-phase carrier using dNTP-3’-R as the raw material; (2) Use TdT to couple ddNTP with -OH at the 3’ end of the oligonucleotide chain that has not undergone base extension to achieve a capping reaction; (3) Then perform deprotection to deprotect the -R at the 3' end of the oligonucleotide chain where base extension occurs to form activated -OH; (4) Repeat steps (1) to (3) until the synthesis of the desired oligonucleotide chain is achieved. 2. The method of claim 1, wherein the ddNTP includes ddATP, ddTTP, ddCTP, ddGTP and ddUTP. 3. The method of claim 1, wherein the dNTP-3'-R includes dNTP-3'ONH ₂ , dNTP-3'-OCN ₃ and dNTP-3'-Aep. 4. The method of claim 1 or 2, wherein the TdT in step (2) is selected from wild-type TdT or E-ZaTdT. 5. The method of claim 1, wherein the oligonucleotide chain is single-stranded DNA, double-stranded DNA or single-stranded RNA. 6. The method of claim 5, wherein the oligonucleotide chain is single-stranded DNA. 7. The method of claim 6, wherein the base extension in step (1) is DNA single base extension based on E-ZaTdT. 8. The method of claim 1, wherein the deprotection in step (2) is performed in an acidic sodium nitrite solution. 9. The method of claim 8, wherein the sodium nitrite solution contains a non-ionic surfactant. 10. The method of claim 9, wherein the nonionic surfactant is Triton X-100 or Tween.