A method for synthesizing ribonucleic acid chains containing non-natural bases by template-free enzymatic method and its application

Abstract

The present invention discloses a method for synthesizing a ribonucleic acid chain containing a non-natural base by a template-free enzymatic method and its application. A natural RNA initiation chain is mixed with a Poly(U) polymerase reaction buffer, and a ribonucleoside triphosphate containing a non-natural base is used as a substrate. Under the catalysis of the Poly(U) polymerase or its mutant, an RNA chain containing a non-natural base at the 3' end is obtained through an enzymatic reaction. The 3' end contains a non-natural base derivative TPT3 Al The RNA chain of TP can then continue to undergo click reactions with azide-modified fluorescent or other functional groups such as N3‑AF488 to achieve orthogonal labeling of RNA fluorescence or other functions. This method has high efficiency, low cost, easy operation, few by-products, and no polluting waste in synthesizing RNA containing non-natural bases. It has potential application value in the enzymatic synthesis of RNA chains containing non-natural bases and functional RNA end labeling.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a method for synthesizing a ribonucleic acid (RNA) chain containing non-natural bases without template by using Poly (U) polymerase wild type (PUP WT) and its mutants PUP H336R and PUP (H336R, N171A, T172S) and its application. Background Art

[0002] RNA is a genetic information carrier present in biological cells and some viruses or viroids, and has multiple biological functions in gene encoding, decoding, regulation and expression. RNA drugs have shown good application prospects in disease treatment, and RNA-based disease prevention, diagnosis and treatment have also rapidly emerged. The application scenarios of RNA are very broad, mainly including RNA vaccines for disease prevention; RNA probes for disease diagnosis; RNA drugs for disease treatment, including siRNA, mRNA drugs, RNA aptamers and antisense oligonucleotide (ASO) drugs. In the field of molecular biology, catalytically active RNA, namely ribozymes, can be used as biocatalysts to degrade specific mRNA sequences and participate in RNA processing and maturation. Guide RNA plays an important role in the CRISPR-Cas9 system, guiding the Cas protein to recognize DNA sequences for cutting and editing.

[0003] The development of non-natural nucleic acids has greatly increased the types of genetic information carriers and expanded the scope of genetic materials. The development of non-natural bases has further expanded the genetic information capacity of nucleic acids and introduced new properties and functions to nucleic acids. At present, the replication, transcription, reverse transcription and translation of non-natural base pairs have been achieved using different polymerases. In addition, the introduction of non-natural bases into organisms has created semi-synthetic organisms that can replicate, transcribe and translate non-natural base pairs, greatly expanding the genetic alphabet in vivo. Non-natural bases are also widely used in vitro, and can be used for site-specific labeling of nucleic acid molecules, coupling with small molecules, and developing new nucleic acid aptamers and nucleic acid aptamer-coupled drugs. Among them, the most representative non-natural base pair is the NaM-TPT3 base pair based on hydrophobic interaction. NaM-TPT3 and its analogs are the only non-natural base pairs that have been successfully used for genetic alphabet expansion, replication, transcription and translation in vivo. The applications of RNA containing non-natural bases mainly include: encoding non-natural amino acids; site-specific orthogonal labeling of RNA, used as a tool for RNA structure and dynamics research; fluorescent labeling of RNA, as a tool for RNA in vivo visualization; site-specific coupling of various functional groups on RNA molecules to prepare coupled drugs or couple GuideRNA and Cas.

[0004] At present, the traditional solid-phase phosphoramidite synthesis method is the most commonly used method for synthesizing RNA chains from scratch. However, it has the disadvantages of requiring unstable phosphoramidites and nucleic acid solid-phase synthesizers, low coupling efficiency, short synthesis sequences, high error rates, and large environmental pollution caused by the organic reagents used. Compared with traditional chemical methods, enzymatic synthesis has great potential to provide high-quality RNA, which can improve the yield and purity of RNA synthesis, simplify downstream purification, eliminate large-scale consumption of organic solvents, avoid the production of toxic byproducts, and reduce the impact of RNA production on the environment. Existing literature shows that Poly(U) polymerase or its mutants can synthesize natural RNA and RNA oligonucleotide chains with modifications on ribose under template-free conditions (DOI: 10.1101 / 2023.06.29.547106), but there is no research on the enzymatic synthesis of RNA containing orthogonal paired non-natural bases. The technology of enzymatic synthesis of RNA containing non-natural bases is of great significance in the efficient preparation of non-natural RNA, RNA labeling based on non-natural base derivatives, the development of novel non-natural nucleic acid aptamers, non-natural nucleic acid drugs and novel RNA vaccines. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings and deficiencies of solid phase synthesis of RNA chains containing non-natural bases and to provide a method for preparing RNA chains containing non-natural bases based on Poly(U) polymerase and its mutants.

[0006] The present invention also aims at using Poly(U) polymerase and its mutants to synthesize RNA chains containing non-natural bases without template in RNA labeling.

[0007] In the present invention, the natural RNA initiation chain is mixed with the Poly(U) polymerase reaction buffer, and a ribonucleoside triphosphate NaMTP, TPT3TP or a TPT3TP derivative (TPT3 Al TP), followed by the addition of Poly(U) polymerase or its mutant, and incubation at 37°C to obtain an RNA chain containing a non-natural base at the 3' end through an enzymatic reaction. Al The RNA chain of TP can then continue to undergo click reaction with azide-modified fluorescent groups such as N3-AF488 to achieve fluorescent labeling of RNA.

[0008] In the present invention, the wild-type Poly(U) polymerase is hereinafter referred to as Poly(U) polymerase PUP WT. The Poly(U) polymerase with a mutation site of H336R is hereinafter referred to as Poly(U) polymerase PUP H336R. The Poly(U) polymerase with mutation sites of H336R, N171A and T172S is hereinafter referred to as PUP(H336R, N171A, T172S), where H336R, N171A, and T172S in brackets represent three mutation sites.

[0009] The purpose of the present invention is achieved through the following steps:

[0010] The method for synthesizing RNA containing non-natural bases without template using Poly(U) polymerase PUP WT, PUP H336R and PUP (H336R, N171A, T172S) comprises the following steps:

[0011] (1) Mix the RNA priming chain and 10×Poly(U) polymerase reaction buffer evenly to obtain a mixed solution 1;

[0012] (2) Adding the mixed solution 1 of step (1) to a ribonucleoside triphosphate containing a non-natural base, NaMTP, TPT3TP or TPT3 Al Any one of TP is used as a substrate and mixed evenly to obtain a mixed solution 2;

[0013] (3) adding Poly(U) polymerase: PUP WT, PUP H336R or PUP (H336R, N171A, T172S) to the mixed solution 2 of step (2) to obtain a mixed solution 3;

[0014] (4) incubating the mixed solution 3 described in step (3) to obtain a mixed solution 4 containing RNA chains containing non-natural bases;

[0015] (5) Adding a loading buffer to the mixed solution 4 in step (4), and heating the mixture to completely denature the RNA chain containing the non-natural base; and analyzing the effect of Poly(U) polymerase in adding the non-natural base ribonucleotide to the 3' end of the RNA chain by using 8M urea denaturing PAGE gel.

[0016] Furthermore, the Poly(U) polymerase reaction buffer in step (1) contains 50 mM sodium chloride, 10 mM Tris-HCl, 10 mM magnesium chloride, and 1 mM dithiothreitol.

[0017] Furthermore, the 10×Poly(U) polymerase reaction buffer in step (1) contains 500 mM sodium chloride, 100 mM Tris-HCl, 100 mM magnesium chloride, and 10 mM dithiothreitol.

[0018] Furthermore, the pH of the Poly(U) polymerase reaction buffer in step (1) is 7-8.

[0019] Furthermore, the pH of the 10×Poly(U) polymerase reaction buffer in step (1) is 7-8.

[0020] Furthermore, the final concentration of the single-stranded RNA in step (1) is 20-200 nM; the final concentration of the 10×Poly (U) polymerase reaction buffer in step (1) is 1×.

[0021] Furthermore, in step (2), the ribonucleoside triphosphate containing non-natural bases NaMTP, TPT3TP or TPT3 Al The final concentration of any one of the TPs is 10-200 μM; the final concentration of the Poly(U) polymerase in step (3) is 0.1-2 μM.

[0022] Furthermore, the incubation temperature in step (4) is 37° C., and the incubation time is 10-60 minutes (depending on the specific practical purpose, the reaction time can be longer).

[0023] Furthermore, the temperature of the heating treatment in step (5) is 92-98° C., and the time is 2-10 minutes.

[0024] Based on the above Poly(U) polymerase and its mutants, the ribonucleoside triphosphate TPT3TP derivative TPT3 was integrated Al TP to initiate the reaction at the 3' end of the RNA chain, and developed a method to utilize Poly(U) polymerase and its mutants to utilize nucleoside triphosphate TPT3 containing functionalized non-natural bases under template-free conditions. Al TP is a technique for orthogonally labeling RNA strands at the 3' end.

[0025] The RNA labeling technique of the present invention is achieved by the following steps:

[0026] (1) Mix the single-stranded RNA primer chain and 10×Poly(U) polymerase reaction buffer evenly to obtain a mixed solution 1;

[0027] (2) Adding the mixed solution 1 of step (1) to a ribonucleoside triphosphate containing a non-natural base, a derivative of TPT3TP, TPT3 Al TP was used as substrate and mixed well to obtain mixed solution 2;

[0028] (3) adding Poly(U) polymerase: PUP WT, PUP H336R or PUP (H336R, N171A, T172S) to the mixed solution 2 of step (2) to obtain a mixed solution 3;

[0029] (4) Incubate the mixed solution 3 in step (3), and purify the product after incubation to obtain 3'

[0030] A mixture 4 of RNA strands having functionalized non-natural bases at their ends;

[0031] (5) copper sulfate and tris(3-hydroxypropyltriazinemethyl)amine (THPTA) were fully mixed at a molar ratio of 5:1 to obtain a mixed solution 5;

[0032] (6) The mixed solution 4 in step (4) is fully mixed with the azide-modified fluorescent molecule N3-AF488 to obtain a mixed solution 6;

[0033] (7) fully mixing the mixed solution 6 described in step (6) with the mixed solution 5 described in step (5) to obtain a mixed solution 7;

[0034] (8) adding ascorbic acid to the mixed solution 7 described in step (7) to obtain a mixed solution 8;

[0035] (9) Purifying the mixed solution 8 described in step (8), adding a loading buffer to the purified product, and heating it to completely denature the RNA chain labeled with the non-natural base derivative; observing the effect of Poly(U) polymerase and its mutant strains on labeling the RNA chain with the non-natural base derivative by using 8M urea denaturing PAGE gel.

[0036] Furthermore, in step (5), the concentrations of copper sulfate and THPTA in the final reaction system are 500 μM and 100 μM, respectively.

[0037] Furthermore, the concentration of the azide-modified fluorescent molecule N3-AF488 in the final reaction system in step (6) is 1 μM.

[0038] Furthermore, the concentration of ascorbic acid in the final reaction system in step (8) is 2.5 mM.

[0039] The other reaction conditions are the same as those for the template-free synthesis of RNA chains containing non-natural bases by Poly(U) polymerase and its mutants.

[0040] Compared with the prior art, the present invention has the following advantages and effects:

[0041] (1) The method provided by the present invention utilizes Poly(U) polymerase and its mutants to integrate ribonucleotides containing non-natural bases into the 3' end of single-stranded RNA to enzymatically synthesize RNA containing non-natural bases.

[0042] (2) This method is used to synthesize RNA chains containing non-natural bases. The synthesis reaction is efficient, the reaction conditions are mild, the cost is low, the operation is convenient, there are few by-products, and no polluting waste is generated. It only requires stable and commonly used basic reagents such as ribonucleoside triphosphate substrates containing non-natural bases, single-stranded RNA initiator chains, and enzyme preparations, and can be completed using the most basic laboratory equipment.

[0043] (3) The RNA chains prepared by this method have great potential application value, such as enzymatic synthesis of RNA, translation of non-natural amino acids, RNA structure research, preparation of functional RNA containing non-natural bases (such as aptamers, ribozymes, siRNA, antisense nucleic acid drugs, etc.) and orthogonal labeling of RNA ends. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is the chemical structure of natural ribonucleoside triphosphates and ribonucleoside triphosphates containing non-natural bases and their derivatives. Base represents adenine, uracil, cytosine or guanine.

[0045] Figure 2 It is a schematic diagram of the synthesis of RNA chains containing non-natural bases catalyzed by Poly(U) polymerase or its mutants.

[0046] Figure 3 This is a graph showing the electrophoresis results of the products of the Poly(U) polymerase and its mutants catalyzing the synthesis of RNA containing the non-natural base NaM in Example 3.

[0047] Figure 4 This is a graph showing the electrophoresis results of the products of the Poly(U) polymerase and its mutants catalyzing the synthesis of RNA containing the non-natural base TPT3 in Example 4.

[0048] Figure 5 The poly (U) polymerase and its mutants in Example 5 are added with a functionalized non-natural base TPT3 at the 3' end of the RNA priming strand. Al The electrophoresis results of the products of ribonucleotides.

[0049] Figure 6 The mutant enzyme PUP H336R of Poly(U) polymerase used in Example 6 to promote the synthesis of TPT3 Al Electrophoresis results of orthogonal fluorescent labeled products of ribonucleotide RNA.

[0050] Figure 7 This is a graph showing the electrophoresis results of the product of the hybrid RNA containing two non-natural base ribonucleotides synthesized by using the mutant enzyme PUP H336R of Poly(U) polymerase in Example 7. DETAILED DESCRIPTION

[0051] The present invention is further described in detail below in conjunction with examples and drawings, but the embodiments of the present invention are not limited thereto. It should be noted that if there are any details not particularly described below, they can be realized or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0052] The ribonucleoside triphosphates containing non-natural bases used in the following examples include NaMTP, TPT3TP and its derivative TPT3 Al TP was purchased from WuXi AppTec.

[0053] The single-stranded RNA sequences used in the examples are shown in Table 1 below:

[0054] Table 1. Natural single-stranded RNA sequences

[0055]

[0056] Example 1. Construction of expression strains of Poly(U) polymerase and its mutants

[0057] The gene fragment of Poly(U) polymerase PUP WT cloned in pET28a(+) plasmid was synthesized by GenScript Biotech. HSDNA polymerase, and conventional overlap extension PCR was performed for site-directed mutagenesis using the Poly(U)WT gene as a template to obtain the PUP H336R gene and the PUP (H336R, N171A, T172S) gene. The primers used for site-directed mutagenesis are shown in Table 2.

[0058] Table 2. Primer sequences required for site-directed mutagenesis

[0059]

[0060] The obtained mutant target gene was cloned into the pET28a(+) plasmid, and the correct recombinant plasmids pET28a(+)-PUP WT, pET28a(+)-PUP H336R and pET28a(+)-PUP(H336R, N171A, T172S) were transformed into the Escherichia coli expression strain BL21(DE3), respectively, to obtain the recombinant expression strains BL21(DE3) / PUP WT, BL21(DE3) / PUPH336R and BL21(DE3) / PUP(H336R, N171A, T172S).

[0061] Example 2. Expression and purification of Poly(U) polymerase and its mutants

[0062] Monoclonal strains BL21(DE3) / PUP WT, BL21(DE3) / PUP H336R and BL21(DE3) / PUP(H336R,N171A,T172S) were selected and cultured in 20 mL of 2×YT medium containing 50 μg / mL kanamycin, shaking at 37°C and 220 rpm overnight. The culture was transferred to 1 L of 2×YT medium containing 50 μg / mL kanamycin for expansion and cultured at 37°C and 220 rpm until OD 600 =0.6-0.8, add 1mM isopropylthiogalactoside, and induce culture overnight at 15°C. After centrifugation at 4°C and 6000 rpm for 10 minutes, collect the cells and resuspend them in 1×Binding Buffer (50mM Tris-HCl, 150mM sodium chloride, 5mM imidazole, 0.1mM ethylenediaminetetraacetic acid, 0.1% Triton X-100). After the cells are broken by high-pressure homogenizer, centrifuge at 4°C and 10000 rpm for 30 minutes, and the supernatant is filtered with 0.45μm and 0.22μm filters to remove cell debris, and the filtered supernatant is purified by nickel column affinity chromatography. After the protein supernatant was incubated with the nickel column, the target protein was eluted with 1×Elution Buffer (50mM Tris-HCl, 150mM sodium chloride, 10-500mM imidazole, 0.1mM ethylenediaminetetraacetic acid, 0.1% Triton X-100, 1mM dithiothreitol, pH 7.5) containing different concentrations of imidazole. The obtained product was verified by SDS-PAGE (polyacrylamide gel electrophoresis). The target protein was further dialyzed and concentrated using a 50kDa Amicon-Ultra centrifugal ultrafiltration tube. The dialyzed and concentrated proteins PUP WT, PUP H336R and PUP (H336R, N171A, T172S) were added with an equal volume of 80% sterile glycerol, and the concentration was detected using a BCA protein detection kit and then stored at -20°C.

[0063] Example 3. Enzymatic preparation of RNA chains containing unnatural base NaM

[0064] (1) 200 nM RNA primer chain FAM-R20 and 1×Poly(U) polymerase reaction buffer, the pH of which is 7.9, are mixed to obtain a mixed solution 1;

[0065] (2) adding NaMTP at a final concentration of 10 μM to the mixed solution 1 of step (1) to obtain a mixed solution 2;

[0066] (3) taking three portions of the mixed solution 2 described in step (2), adding 1 μM PUP WT, PUP H336R or PUP (H336R, N171A, T172S) respectively to obtain three portions of mixed solution 3;

[0067] (4) incubating the mixed solution 3 in step (3) at 37° C. for 30 minutes to obtain a mixed solution 4;

[0068] (5) Add 2×TBE-Urea loading buffer to the mixed solution 4 in step (4), and heat it at 95° C. for 10 minutes to completely denature the RNA; analyze the effect of Poly(U) polymerase and its mutants in adding ribonucleotides containing non-natural bases to the 3' end of the RNA chain by denaturing PAGE gel electrophoresis.

[0069] The electrophoresis results are as follows Figure 3 As shown: Figure 3 Lane 1 is the RNA priming chain control, lane 2 is the product of polymerase PUP WT integrating NaMTP at the 3' end of RNA to synthesize RNA containing the non-natural base NaM; lane 3 is the product of polymerase PUP H336R integrating NaMTP at the 3' end of RNA to synthesize RNA containing the non-natural base NaM; lane 4 is the product of polymerase PUP (H336R, N171A, T172S) integrating NaMTP at the 3' end of RNA to synthesize RNA containing the non-natural base NaM. Figure 3 It can be seen that Poly(U) polymerase and its mutants can incorporate at most one NaM ribonucleotide at the 3' end of RNA.

[0070] Example 4. Enzymatic preparation of RNA chains containing the unnatural base TPT3

[0071] The steps are the same as those in Example 3, wherein TPT3TP is added in step (2).

[0072] The electrophoresis results are as follows Figure 4 As shown: Figure 4 Lane 1 is the RNA priming chain control, lane 2 is the product of polymerase PUP WT integrating TPT3TP at the 3' end of RNA to synthesize RNA containing the unnatural base TPT3; lane 3 is the product of polymerase PUP H336R integrating TPT3TP at the 3' end of RNA to synthesize RNA containing the unnatural base TPT3; lane 4 is the product of polymerase PUP (H336R, N171A, T172S) integrating TPT3TP at the 3' end of RNA to synthesize RNA containing the unnatural base TPT3. Figure 4It can be seen that the integration activity of PUPH336R for TPT3 is significantly higher than that of PUP WT and PUP (H336R, N171A, T172S). Under the present implementation conditions, PUP H336R can integrate up to about 10 TPT3 ribonucleotides at the 3' end of RNA.

[0073] Example 5. Enzymatic addition of the non-natural base derivative TPT3 to the 3' end of the RNA priming strand Al Ribonucleotide

[0074] The steps are the same as those in Example 3, except that TPT3 is added in step (2). Al TP.

[0075] The electrophoresis results are as follows Figure 5 As shown: Figure 5 Lane 1 is the RNA priming strand control, and lane 2 is the polymerase PUP WT incorporating TPT3 at the 3' end of the RNA. Al TP, synthetic TPT3 containing unnatural base Al Lane 3 shows the product of RNA; Lane 3 shows the polymerase PUP H336R integrating TPT3 at the 3' end of RNA Al TP, synthetic TPT3 containing unnatural base Al Lane 4 shows the product of RNA polymerase PUP (H336R, N171A, T172S) integrating TPT3 at the 3' end of RNA Al TP, synthetic TPT3 containing unnatural base Al The product of RNA. Figure 5 It can be seen that PUP H336R is effective for TPT3 Al The integration activity of TP was higher than that of PUP WT and mutant PUP (H336R, N171A, T172S). Under the conditions of this implementation, PUP H336R could integrate up to 5 TPT3 at the 3' end of RNA. Al ribonucleotides, with two TPT3s integrated at the 3' end Al Ribonucleotides are the main products.

[0076] Example 6. Using Poly(U) polymerase and a derivative of the unnatural base ribonucleoside triphosphate TPT3TP Al TP functionally labels RNA (taking fluorescent labeling as an example)

[0077] (1) 200 nM RNA initiator chain R30 and 1×Poly(U) polymerase reaction buffer, wherein the pH of the Poly(U) polymerase reaction buffer is 7.9, are mixed uniformly to obtain a mixed solution 1;

[0078] (2) Add TPT3 to a final concentration of 10 μM into the mixed solution 1 of step (1) Al TP, to obtain mixed solution 2;

[0079] (3) adding 1 μM polymerase PUP H336R to the mixed solution 2 of step (2) to obtain a mixed solution 3;

[0080] (4) incubating the mixed solution 3 in step (3) at 37° C. for 30 minutes, and purifying the product using a Zymo ssDNA / RNAClean&Concentrator kit to obtain a mixed solution 4 containing an RNA chain with a functionalized non-natural base at the 3′ end;

[0081] (5) copper sulfate and THPTA were fully mixed at a molar ratio of 5:1 to obtain a mixed solution 5;

[0082] (6) in step (4), the mixed solution 4 is fully mixed with 1 μM azide-modified fluorescent molecule N3-AF488 to obtain a mixed solution 6;

[0083] (7) Fully mixing the mixed solution 6 described in step (6) with the mixed solution 5 described in step (5) with a final concentration of 500 μM:100 μM to obtain a mixed solution 7;

[0084] (8) adding 2.5 mM ascorbic acid to the mixed solution 7 in step (7), and reacting at room temperature for 10 minutes to obtain a mixed solution 8;

[0085] (9) The mixed solution 8 described in step (8) is purified using a Zymo ssDNA / RNA Clean&Concentrator kit to obtain a mixed solution 9.

[0086] (9) Add 2×TBE-Urea loading buffer to the mixed solution 9 in step (8), heat it at 95° C. for 10 minutes to denature the RNA chain; observe the activity of Poly(U) polymerase through the non-natural base TPT3 derivative TPT3 by 8 M urea denaturing PAGE gel electrophoresis. Al Results of RNA labeling.

[0087] The electrophoresis results are as follows Figure 6 As shown: Figure 6 Lane 1 is the RNA priming strand control; Lane 2 is the polymerase PUP H336R integrating TPT3 at the 3' end of RNA Al TP, synthetic TPT3 containing unnatural base Al Lane 3 is the product of RNA with unnatural base TPT3 at the 3' end AlThe labeled RNA reacts with the azide-modified fluorophore N3-AF488 to prepare the fluorescent labeled RNA product. Before RNA staining, the TPT3 at the 3' end can be directly observed under blue light. Al RNA coupled with the azide-modified fluorophore N3-AF488 can be observed only after staining, which indicates that the present invention is effective in functional orthogonal labeling of RNA.

[0088] Example 7

[0089] (1) 200 nM RNA primer chain FAM-R20 and 1×Poly(U) polymerase reaction buffer, the pH of which is 7.9, are mixed to obtain a mixed solution 1;

[0090] (2) adding NaMTP at a final concentration of 10 μM to the mixed solution 1 of step (1) to obtain a mixed solution 2;

[0091] (3) taking three portions of the mixed solution 2 described in step (2), adding 1 μM PUP WT, PUP H336R or PUP (H336R, N171A, T172S) respectively to obtain three portions of mixed solution 3;

[0092] (4) incubating the mixed solution 3 in step (3) at 37° C. for 30 minutes to obtain a mixed solution 4;

[0093] (5) adding TPT3TP with a final concentration of 10 μM to the mixed solution 4 in step (4) to obtain a mixed solution 5;

[0094] (6) incubating the mixed solution 5 in step (5) at 37° C. for 30 minutes to obtain a mixed solution 6;

[0095] (7) Add 2×TBE-Urea loading buffer to the mixed solution 6 in step (6), and heat it at 95° C. for 10 minutes to completely denature the RNA; analyze the effect of Poly(U) polymerase and its mutants in adding two ribonucleotides containing non-natural bases to the 3' end of the RNA chain by denaturing PAGE gel electrophoresis.

[0096] The electrophoresis results are as follows Figure 7 As shown: Figure 7Lanes 1 and 4 are RNA priming chain controls, lane 2 is polymerase PUPWT integrating NaMTP at the 3' end of RNA to synthesize RNA products containing the non-natural base NaM; lane 3 is polymerase PUP WT integrating TPT3TP at the 3' end of RNA containing the non-natural base NaM to synthesize hybrid RNA products containing the non-natural bases NaM and TPT3; lane 5 is polymerase PUP H336R integrating NaMTP at the 3' end of RNA to synthesize RNA products containing the non-natural base NaM; lane 6 is polymerase PUP H336R integrates TPT3TP at the 3' end of RNA containing the unnatural base NaM to synthesize a hybrid RNA product containing the unnatural bases NaM and TPT3; Lane 7 shows that polymerase PUP (H336R, N171A, T172S) integrates NaMTP at the 3' end of RNA to synthesize a RNA product containing the unnatural base NaM; Lane 8 shows that polymerase PUP (H336R, N171A, T172S) integrates TPT3TP at the 3' end of RNA containing the unnatural base NaM to synthesize a hybrid RNA product containing the unnatural base NaM and TPT3. Figure 7 It can be seen that Poly(U) polymerase and its mutants can integrate two ribonucleotides, NaM and TPT3, at the 3' end of RNA to synthesize a hybrid non-natural base ribonucleotide chain.

Claims

1. A method for synthesizing a ribonucleic acid chain containing a non-natural base by a template-free enzymatic method, characterized in that: The following steps are involved: (1) mixing the natural RNA priming chain and the Poly(U) polymerase reaction buffer evenly to obtain a mixed solution 1; (2) Adding a ribonucleoside triphosphate containing a non-natural base, TPT3TP, NaMTP or TPT3 Al Any one of TP, to obtain mixed solution 2; (3) adding a wild-type Poly(U) polymerase, a Poly(U) polymerase with a mutation site of H336R, or a Poly(U) polymerase with mutation sites of H336R, N171A and T172S to the mixed solution 2 obtained in step (2) to obtain a mixed solution 3; (4) incubating the mixed solution 3 obtained in step (3) to obtain a mixed solution 4 containing an RNA chain containing a non-natural base at the 3' end; (5) adding a loading buffer to the mixed solution 4 obtained in step (4), heating the mixture to completely denature the RNA chain containing the non-natural base, and analyzing the effect of adding the non-natural base to the 3' end of the RNA chain by the wild-type Poly(U) polymerase, the Poly(U) polymerase with the mutation site H336R, or the Poly(U) polymerase with the mutation sites H336R, N171A and T172S using a denaturing gel; The sequence of the wild-type Poly (U) polymerase is: MNISSAQFIPGVHTVEEIEAEIHKNLHISKSCSYQKVPNSHKEFTKFCYEVYNEIKISDKEFKEKRAALDTLRLCLKRISPDAELVAFGSLESGLALKNSDMDLCVLMDSRVQSDTIALQFYEELIAEGFEGKFLQRARIPIIKLTSDTKNGFGASFQCDIGFNNRLAIHNTLLLSSYTKLDARLKPMVLLVK HWAKRKQINSPYFGTLSSYGYVLMVLYYLIHVIKPPVFPNLLLSPLKQEKIVDGFDVGFDDKLEDIPPSQNYSSLGSLLHGFFRFYAYKFEPREKVVTFRRPDGYLT KQEKGWTSATEHTGSADQIIKDRYILAIEDPFEISHNVGRTVSSSGLYRIRGEFMAASRLLNSRSYPIPYDSLFEEAPIPPRRQKKTDEQSNKKLLNETDGDNSEKL.

2. The method for synthesizing a ribonucleic acid chain containing a non-natural base by a template-free enzymatic method according to claim 1, characterized in that: In step (1), the Poly(U) polymerase reaction buffer contains 40-60 mM sodium chloride, 5-15 mM Tris-HCl, 5-15 mM magnesium chloride and 0.5-1.5 mM dithiothreitol.

3. The method for synthesizing a ribonucleic acid chain containing a non-natural base by a template-free enzymatic method according to claim 1, characterized in that: In step (1), the Poly(U) polymerase reaction buffer contains 50 mM sodium chloride, 10 mM Tris-HCl, 10 mM magnesium chloride and 1 mM dithiothreitol.

4. The method for synthesizing a ribonucleic acid chain containing a non-natural base by a template-free enzymatic method according to claim 1, characterized in that: In step (1), the pH of the Poly(U) polymerase reaction buffer is 7-8; the final concentration of the natural RNA initiator chain is 20-200 nM; and the final concentration of the Poly(U) polymerase reaction buffer is 1×.

5. The method for synthesizing a ribonucleic acid chain containing a non-natural base by a template-free enzymatic method according to claim 1, characterized in that: In step (2), the ribonucleoside triphosphate containing non-natural bases TPT3TP, NaMTP or TPT3 Al The final concentration of TP is 10-200 μM.

6. The method for synthesizing a ribonucleic acid chain containing a non-natural base by a template-free enzymatic method according to claim 1, characterized in that: In step (3), the final concentration of the wild-type Poly(U) polymerase, the Poly(U) polymerase with the mutation site H336R, or the Poly(U) polymerase with the mutation sites H336R, N171A and T172S is 0.1-2 μM.

7. The method for synthesizing a ribonucleic acid chain containing a non-natural base by a template-free enzymatic method according to claim 1, characterized in that: In step (4), the incubation temperature is 37°C and the incubation time is 10-60 minutes.

8. The method for synthesizing a ribonucleic acid chain containing a non-natural base by a template-free enzymatic method according to claim 1, characterized in that: In step (5), the temperature of the heating treatment is 92-98° C.; the time of the heating treatment in step (5) is 2-15 minutes.

9. Use of a ribonucleic acid RNA chain containing a non-natural base synthesized by the method according to any one of claims 1 to 8 in RNA labeling, characterized in that: The steps include: (1) Mix the single-stranded RNA primer chain and 10× Poly(U) polymerase reaction buffer evenly to obtain a mixed solution 1; (2) Adding the mixed solution 1 of step (1) to the ribonucleoside triphosphate containing non-natural bases, TPT3TP derivative TPT3 Al TP was used as substrate and mixed well to obtain mixed solution 2; (3) Adding Poly(U) polymerase: PUP WT, PUP H336R or PUP (H336R, N171A, T172S) to the mixed solution 2 in step (2) to obtain a mixed solution 3; (4) incubating the mixed solution 3 in step (3), and purifying the product after incubation to obtain a mixed solution 4 containing an RNA chain having a functionalized non-natural base at the 3' end; (5) copper sulfate and tris(3-hydroxypropyltriazinemethyl)amine (THPTA) are mixed at a molar ratio of 5:1 to obtain a mixed solution 5; (6) The mixed solution 4 in step (4) is fully mixed with the azide-modified fluorescent molecule N3-AF488 to obtain a mixed solution 6; (7) Fully mixing the mixed solution 6 described in step (6) with the mixed solution 5 described in step (5) to obtain a mixed solution 7; (8) adding ascorbic acid to the mixed solution 7 of step (7) to obtain a mixed solution 8; (9) Purifying the mixed solution 8 described in step (8), adding loading buffer to the purified product, and heating it to completely denature the RNA chain labeled with the non-natural base derivative; observing the effect of Poly(U) polymerase and its mutant strains on labeling RNA chains with non-natural base derivatives through 8M urea denaturing PAGE gel.

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