RNA acylation modification method and application thereof

Abstract

The invention provides an RNA acylation modification method and application thereof, and relates to the field of RNA in-vitro chemical modification. The invention constructs a universal method for RNA acylation modification guided by a nucleic acid template. An RNA molecule with acylated hydroxyl groups of ribose at the 3'end is generated by using a DNA template molecule and a donor peptide nucleic acid (PNA) molecule. The RNA acylation modification method guided by the nucleic acid template has the advantages of simplicity in operation, wide substrate application range and site specificity, and the generated acylated RNA molecule is beneficial to in-vivo and in-vitro related RNA molecular therapy and construction of an acellular in-vitro expression system and an mRNA display system of non-natural protein or polypeptide; the method is a convenient and powerful universal method for in-vitro fixed-point modification of RNA.

Description

Technical Field

[0001] The present invention relates to the field of RNA in vitro chemical modification, and in particular to a method and application of RNA acylation modification. Background Art

[0002] RNA is a crucial biological macromolecule involved in protein expression and gene regulation. RNA plays a crucial role in the development of biomacromolecule drugs, with over 50 RNA drugs currently approved for disease treatment. In particular, RNA drugs have demonstrated remarkable efficacy in the control and treatment of a wide range of diseases, including mRNA vaccines for infectious diseases and small RNA drugs for tumor immunotherapy. Small RNA drug development is broadly focused, encompassing viral diseases, cardiovascular diseases, tumors, rare diseases, and metabolic disorders. Hundreds of RNA drugs are currently in clinical development, and it is foreseeable that a significant number will be approved for marketing in the future.

[0003] In biomedical applications, two important issues that need to be addressed in the development of RNA drugs are how to improve their in vivo stability and tissue-specific delivery. Chemical modification of RNA is an important strategy to address the in vivo stability and tissue-specific delivery of RNA drugs: chemically modifying or transforming RNA molecules to make them stable in vivo and evade recognition by the immune system; and introducing special chemical modifications to achieve tissue-specific delivery of RNA drugs. How different chemical modifications affect the enzymatic stability, metabolic stability, and tissue specificity of RNA molecules still urgently needs to be explored. The bottleneck that limits the study of the biological functions of chemically modified RNA is the difficulty in obtaining RNA samples modified at specific sites. Therefore, developing a simple method for in vitro chemical modification of RNA is an important core technology in the field of RNA drug research and development.

[0004] At present, the main methods for RNA acylation modification include de novo chemical synthesis, RNA semi-synthesis using ligase, and post-modification of expressed RNA. In principle, de novo chemical synthesis can introduce various modifications at any site of RNA, but it involves tedious multi-step chemical synthesis, is costly, and is not suitable for the preparation of long-chain RNA. RNA semi-synthesis using ligase can obtain long-chain chemically modified RNA, but it still involves tedious chemical synthesis and modification of RNA molecules. Post-modification of expressed RNA uses highly active oxygen esters to acylate the 2' hydroxyl group of RNA, without the need for tedious chemical synthesis and modification of RNA molecules. Kool et al. from Stanford University in the United States developed a series of active small molecules that can react with the 2' hydroxyl group of RNA molecules. It should be pointed out that this post-modification of expressed RNA has the following defects: (1) It is difficult to achieve modification of hydroxyl groups at specific sites of RNA; (2) The active small molecules have a simple structure and are limited in variety.

[0005] Suga et al. at the University of Tokyo in Japan developed a tRNA acylation method based on flexizyme nucleases, enabling the preparation of hundreds of unnatural aminoacylated tRNAs and establishing a robust RaPID screening system for non-classical cyclic peptides. RNA acylation by nucleases is limited to tRNA modification and is unsuitable for post-modification of other types of RNA. Using aminoacyl-tRNA synthetases is also an effective method for tRNA post-modification. However, this method generally limits the types of amino acids that can be introduced and requires tedious directed enzyme evolution for different molecules.

[0006] DNA-templated chemistry is a unique chemical reaction mechanism that enables rapid and efficient reactions between two reactive groups at extremely low concentrations. It has been fully demonstrated in the construction of molecular libraries and in the in vitro amplification and detection of nucleic acids. It is worth mentioning that Oliver et al. from Humboldt University in Germany have achieved acyl transfer reactions and polypeptide ligation reactions on peptide nucleic acids (PNA) by using DNA or RNA as templates. DNA-templated chemistry has not yet been used for post-modification of RNA. By deeply analyzing the structures of PNA-DNA and PNA-RNA duplexes, a universal method for nucleic acid-templated RNA acylation modification has been developed for the rapid and convenient preparation of acylated RNA molecules, which will establish a powerful universal platform for the preparation of acylated RNA. Summary of the Invention

[0007] In view of the shortcomings of the existing technology, the present invention provides a method for RNA acylation modification and its application, and designs a simple and efficient DNA template-guided RNA acylation modification method for preparing acylated RNA molecules.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions: A method for RNA acylation modification, comprising the following steps: S1. The receptor RNA molecule and the template DNA molecule are hybridized after high-temperature denaturation and low-temperature annealing in a buffered salt solution to form a receptor RNA•template DNA hybrid; S2. Through the principle of complementary base pairing, the donor PNA molecule hybridizes with the above receptor RNA•template DNA hybrid to form a receptor RNA•template DNA•donor PNA double helix; S3. Through the adjacent space effect, the acyl group of the donor PNA molecule on the acceptor RNA, template DNA, and donor PNA double helix is ​​transferred to the hydroxyl group of the 3'-terminal ribose of the acceptor RNA to form a nucleic acid sample; S4. The nucleic acid sample is subjected to the action of deoxyribonuclease DNase I to degrade the template DNA, and then the target acylated RNA molecule is separated.

[0009] Preferably, the ribose at the 3' end of the receptor RNA molecule contains at least one hydroxyl group, and the receptor RNA molecule is any one of messenger RNA, ribosomal RNA, transfer RNA, small nuclear RNA, microRNA, small nucleolar RNA or non-coding RNA in a single-stranded RNA molecule.

[0010] Preferably, the N-terminus of the donor PNA molecule is connected to the acyl group to be transferred via a phenol ester or a thioester, and the donor PNA molecule has the following general formula: where X a For phenolic esters: , Aryl thioesters: or alkyl thioesters: Any one of the following; R is any one of a natural aminoacyl group, a non-natural aminoacyl group, an oligopeptide acyl group composed of natural amino acids, an oligopeptide acyl group containing non-natural amino acids, an acyl group containing a natural or non-natural monosaccharide, an acyl group containing a natural or non-natural polysaccharide, an acyl group containing a peptide nucleic acid or nucleic acid, an acyl group of a natural amino acid protein, an acyl group of a non-natural amino acid protein, or an acyl group containing a lipid; Z is any one of a hydrogen, an electron-donating, or an electron-withdrawing substituent on the benzene ring; X b X is any one of hydrogen, a side chain group of a natural amino acid except glycine and proline, or a side chain group of a non-natural amino acid; c Any one of the four bases of deoxyribonucleic acid; X d Any one of the four bases of deoxyribonucleic acid; X e It is any one of the oligopeptide sequences composed of amino groups, natural or non-natural amino acids; N is nitrogen; H is hydrogen; O is oxygen; S is a sulfur atom; n is any number between 0 and 5; and m is any number between 2 and 49.

[0011] Preferably, the template DNA molecule is designed to simultaneously hybridize with the acceptor RNA and the donor PNA to form a double helix, and the hydroxyl group of the ribose at the 3' end of the acceptor RNA is spatially close to the acylated group connected to the N-terminus of the donor PNA.

[0012] Preferably, the buffered saline solution in step S1 is any one of PBS, HEPES, NaOAc, Tris and imidazole, and has a pH range of 6.5 to 10.0.

[0013] Preferably, in the double helix structure of the acceptor RNA, template DNA, and donor PNA in step S2: there are no other bases between the base paired between the 3' end of the acceptor RNA and the template DNA and the base paired between the N end of the donor PNA and the template DNA, and the hydroxyl group of the ribose at the 3' end of the acceptor RNA is spatially close to the acyl group of the donor PNA; the number of paired bases in the region where the acceptor RNA and the template DNA undergo base complementary pairing is any number between 5 and 500; and the number of paired bases in the region where the donor PNA and the template DNA undergo base complementary pairing is any number between 3 and 50.

[0014] Preferably, the concentration range of the acceptor RNA, template DNA and donor PNA in steps S1, S2 and S3 is 0.001 μM to 100.0 mM, and the reaction time is 5 min to 72 h, and the reaction temperature is 0 to 75°C.

[0015] Preferably, the method for isolating the target acylated RNA molecule in step S4 is any one of high performance liquid chromatography separation, size exclusion chromatography, ethanol precipitation, isopropanol precipitation, phenol-chloroform extraction, nucleic acid purification column, PAGE purification or Trizol extraction.

[0016] The above-mentioned RNA acylation modification method can be applied to RNA interference therapy, antisense oligonucleotides, small activating RNA therapy, messenger RNA-based therapy, mRNA display of non-standard amino acid cyclic peptides, ribosome display, in vitro expression of protein macromolecular drugs, in vitro ribosome translation of non-natural amino acids in cell-free systems, and in vitro genetic codon reprogramming.

[0017] The present invention provides a method for RNA acylation modification and its application, which has the following advantages over the prior art: (1) Compared with de novo chemical synthesis and RNA semisynthesis by ligase, the present invention uses a DNA template to guide the transfer of the acyl group on the donor PNA to the hydroxyl group of the ribose at the 3' end of the RNA. It does not involve the time-consuming and complicated chemical synthesis and modification of RNA molecules, and the operation steps are simpler and easier to implement. (2) Compared with existing post-modification methods for expressed RNA, the present invention can achieve site-specific acylation modification of the 3'-terminal ribose hydroxyl group of RNA and is applicable to the acylation of structurally complex molecules with RNA; (3) Compared with the tRNA modification method of flexizyme nuclease, the present invention is not limited to tRNA and can be applied to various types of RNA molecules, including any one of mRNA, rRNA, snRNA, miRNA, snoRNA or lncRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The general formula for the double helix of acceptor RNA • template DNA • donor PNA; Figure 2 The structural formula a), liquid chromatography b) and mass spectrum c) of the donor PNA1 in Example 1 of the present invention are shown; Figure 3 The structural formula a), liquid chromatography b) and mass spectrum c) of the donor PNA2 in Example 2 of the present invention are shown; Figure 4 The structural formula a), liquid chromatogram b) and mass spectrum c) of the donor PNA3 in Example 3 of the present invention are shown; Figure 5 This is an acidic PAGE gel analysis of the acylation modification of the template DNA1 guiding RNA1 of the present invention. DETAILED DESCRIPTION

[0019] In order to illustrate the present invention more clearly, the following will further illustrate it through implementation cases and drawings.

[0020] The steps included in the present invention are as follows: (1) The RNA acylation modification method includes an acceptor RNA molecule, a donor peptide nucleic acid (PNA) molecule, and a template DNA molecule; The N-terminus of the donor peptide nucleic acid (PNA) molecule is connected to the acyl group to be transferred via a phenol ester or thioester, and the template DNA molecule is designed to simultaneously hybridize with the receptor RNA and the donor PNA to form a double helix, and to spatially bring the hydroxyl group of the ribose sugar at the 3' end of the receptor RNA into close proximity with the acylated group connected to the N-terminus of the donor PNA.

[0021] Based on the above characteristics, three molecules are selected as follows: The receptor RNA1 molecule has the following sequence: 5'-GGUUAGAUUCCCGGGCUUUCCCCA-3'; Template DNA1 molecule, the sequence is as follows: 5'-GATGTTGATTGGCGGAAAGCCCGGGAATCTAACC-3'; The donor PNA molecule has the following structural formula: The receptor RNA molecule and template DNA molecule are both composed of natural nucleotides, with the 5'-hydroxyl group of ribose and deoxyribose at the 5'-end, and the 3'-hydroxyl group of ribose and deoxyribose at the 3'-end, respectively. The donor PNA molecule can be any of PNA1 (phenolic ester), PNA2 (aryl thioester), or PNA3 (alkyl thioester) in the structural formula, and the acyl group on the donor PNA molecule is a Tyr aminoacyl group. The receptor RNA1, template DNA1, and donor PNA1 (or PNA2, or PNA3) provided herein are intended only to illustrate the technical details and specific embodiments of the present invention and do not constitute the entire content of this patent. During DNA-templated RNA acylation, the base sequence and length of the receptor RNA molecule can be modified as needed, and the modified receptor RNA molecule still falls within the scope of protection of this patent. When performing DNA-templated RNA acylation modification, the base sequence, length, and acyl group of the donor PNA molecule can be changed as needed, and a substituent can be introduced into the g-position of the monomer structure of the PNA as needed. The transformed donor PNA molecule still falls within the scope of protection of this patent. When performing DNA-templated RNA acylation modification, the sequence of the template DNA molecule can be changed according to the base sequence of the acceptor RNA molecule and the donor PNA molecule. The transformed template DNA molecule still falls within the scope of protection of this patent. Therefore, changes and modifications to the acceptor RNA molecule, template DNA molecule, and donor PNA molecule made on the basis of this patent still fall within the scope of protection of this patent.

[0022] (2) In a buffered salt solution, the three molecules form a double helix of acceptor RNA, template DNA, and donor PNA through the principle of complementary base pairing, and the acyl group of the donor PNA molecule is transferred to the hydroxyl group of the 3'-terminal ribose of the acceptor RNA.

[0023] The buffer solution is any one of PBS (phosphate), HEPES (4-hydroxyethylpiperazineethanesulfonic acid), NaOAc (sodium acetate), Tris (tris(hydroxymethylaminomethane)), and imidazole, with a pH range of 6.5 to 10.0; the concentration range of the acceptor RNA, template DNA, and donor PNA is 0.001 μM to 100.0 mM, the reaction time is 5 minutes to 72 hours, and the reaction temperature is 0 to 75°C; the general formula of the acceptor RNA•template DNA•donor PNA double helix is ​​as follows: Figure 1 As shown; Figure 1In the present invention, there is no other base between the base paired between the 3' end of the acceptor RNA and the template DNA and the base paired between the N-terminus of the donor PNA and the template DNA, so that the hydroxyl group of the ribose at the 3' end of the acceptor RNA is spatially close to the acyl group of the donor PNA; Region 1 is the region where base complementary pairing occurs between the acceptor RNA and the template DNA, and the number of paired bases is any number between 5 and 500; Region 2 is the region where base complementary pairing occurs between the donor PNA and the template DNA, and the number of paired bases is any number between 3 and 50; all simple changes to the conditions made on the basis of this patent are still within the scope of protection of this patent.

[0024] (3) Under the action of deoxyribonuclease DNase I, the template DNA in the above reaction system is degraded, and then the target acylated RNA molecule is separated.

[0025] Among them, the method for isolating the acylated RNA product is any one of high performance liquid chromatography separation, size exclusion chromatography, ethanol precipitation, isopropanol precipitation, phenol-chloroform extraction, nucleic acid purification column, PAGE purification or Trizol extraction; all simple changes to the conditions made on the basis of this patent are still within the scope of protection of this patent.

[0026] Example 1: According to the above content Chemical synthesis of donor PNA1: 4.3 µmol of Rinkamide MBHA resin (6.5 mg, 0.658 mmol / g) was weighed and transferred to a 1.0 mL-sized syringe with a sieve plate. 0.2 mL of DMF was added and the mixture was swollen at room temperature for 15 min.

[0027] After draining the DMF solution, add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. After draining the 20% piperidine in DMF solution, add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for another 6 minutes. After draining the 20% piperidine in DMF solution, wash the resin thoroughly four times with 0.2 mL of DMF.

[0028] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-C(Bhoc)-OH (2.0 mg), oxyma (0.4 mg), and N,N'-diisopropylcarbodiimide (0.43 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction solution, wash the resin four times with 0.2 mL of DMF.

[0029] Add 0.2 mL of acetic anhydride blocking reagent (DMF: acetic anhydride: 2,6-lutidine = 89:5:6, volume ratio) and incubate the resin at room temperature for 2 min.

[0030] After draining the acetic anhydride blocking reagent, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of a 20% piperidine solution in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protecting group removal procedure. After draining the 20% piperidine solution in DMF, wash the resin four times with 0.2 mL of DMF.

[0031] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-T-OH (3.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0032] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-A(Bhoc)-OH (4.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0033] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-C(Bhoc)-OH (4.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0034] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-A(Bhoc)-OH (4.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0035] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-A(Bhoc)-OH (4.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0036] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-C(Bhoc)-OH (4.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0037] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-T-OH (3.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0038] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-A(Bhoc)-OH (4.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0039] Add 130 µL of DMF containing a condensation reagent containing p-hydroxyphenylacetic acid (3.8 mg, 25 µmol), oxyma (3.6 mg, 25 µmol), and N,N'-diisopropylcarbodiimide (3.9 µL, 25 µmol). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0040] Add 160 µL of DCM / DMF (1:1) condensation reagent containing Boc-Tyr(tBu)-OH (16.8 mg, 50 µmol), 4-dimethylaminopyridine (0.3 mg), and N,N'-diisopropylcarbodiimide (4.0 µL, 25 µmol). Incubate the resin with shaking at room temperature for 16 hours. Wash the resin with DMF and DCM, then air-dry.

[0041] Add 0.5 mL of freshly prepared trifluoroacetic acid lysis buffer (trifluoroacetic acid:phenol:water:triisopropylsilane = 88:5:5:2, v / v). After incubation at room temperature for 2 hours, collect the trifluoroacetic acid lysis buffer, add 8 volumes of ice-cold ether, and centrifuge to obtain crude PNA1. The crude PNA1 was dissolved in 0.1% water. The structural formula, HPLC analysis, and mass spectrometry analysis of PNA1 are shown in Figure 2. Figure 2 As shown, confirm the correctness of the product (molecular formula: C 113 H 137 N 53 O 29 The target PNA1 was purified by semi-preparative chromatography and freeze-dried to obtain a powdered PNA1 molecule (2.0 mg).

[0042] Example 2: According to the above content Chemical synthesis of donor PNA2: 4.3 µmol of Rinkamide MBHA resin (6.5 mg, 0.658 mmol / g) was weighed and transferred to a 1.0 mL-sized syringe with a sieve plate. 0.2 mL of DMF was added and the mixture was swelled at room temperature for 15 minutes.

[0043] After draining the DMF solution, add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. After draining the 20% piperidine in DMF solution, add 0.1 mL of 20% piperidine in DMF and incubate again at room temperature for 6 minutes. After draining the 20% piperidine in DMF solution, wash the resin thoroughly four times with 0.2 mL of DMF.

[0044] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-C(Bhoc)-OH (2.0 mg), oxyma (0.4 mg), and N,N'-diisopropylcarbodiimide (0.43 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction solution, wash the resin four times with 0.2 mL of DMF.

[0045] Add 0.2 mL of acetic anhydride blocking reagent (DMF: acetic anhydride: 2,6-lutidine = 89:5:6, volume ratio) and incubate the resin at room temperature for 2 minutes. After draining the acetic anhydride blocking reagent, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protecting group removal procedure. After draining the 20% piperidine in DMF, wash the resin four times with 0.2 mL of DMF.

[0046] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-T-OH (3.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0047] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-A(Bhoc)-OH (4.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0048] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-C(Bhoc)-OH (4.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0049] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-A(Bhoc)-OH (4.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0050] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-A(Bhoc)-OH (4.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0051] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-C(Bhoc)-OH (4.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0052] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-T-OH (3.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0053] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-A(Bhoc)-OH (4.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes.

[0054] Repeat the Fmoc removal procedure and wash the resin four times with DMF. Add 130 µL of DMF containing condensation reagents containing p-mercaptophenylacetic acid (4.8 mg, 25 µmol), oxyma (4.0 mg), and N,N'-diisopropylcarbodiimide (4.4 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc removal procedure and wash the resin four times with DMF.

[0055] The resin was transferred to a 1.5 mL centrifuge tube and incubated with 1.5 mL of dithiothreitol / N,N-diisopropylaminoethylamine / NMP / water (231 mg / 261 µL / 1.1 mL / 150 µL) at room temperature for 80 minutes. The resin was washed with DMF and then with DCM / DMF (1:1). Then, 160 µL of DCM / DMF (1:1) condensation reagent containing Boc-Tyr(tBu)-OH (16.8 mg, 50 µmol), 4-dimethylaminopyridine (0.3 mg), and N,N'-diisopropylcarbodiimide (4.0 µL) was added.

[0056] The resin was incubated at room temperature with shaking for 14 hours. The resin was washed with DMF and DCM in sequence and air-dried. 0.5 mL of freshly prepared trifluoroacetic acid lysis buffer (trifluoroacetic acid:phenol:water:triisopropylsilane = 88:5:5:2, v / v) was added. After incubation at room temperature for 2 hours, the trifluoroacetic acid lysis buffer was collected, 8 volumes of ice-cold ether were added, and the crude PNA2 was obtained by centrifugation. The crude PNA2 was dissolved in 0.1% water. The structural formula, HPLC analysis, and mass spectrometry analysis of PNA2 are shown in Figure 2. Figure 3 As shown, confirm the correctness of the product (molecular formula: C 113 H 137 N 53 O 28 The target PNA2 was purified by semi-preparative chromatography and freeze-dried to obtain a powdered PNA2 molecule (2.5 mg).

[0057] Example 3: According to the above content Chemical synthesis of donor PNA3 4.3 µmol of Rink amide MBHA resin (6.5 mg, 0.658 mmol / g) was weighed and transferred to a 1.0 mL-sized syringe with a frit. 0.2 mL of DMF was added and the mixture was swollen at room temperature for 15 min.

[0058] After draining the DMF solution, add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. After draining the 20% piperidine in DMF solution, add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for another 6 minutes. After draining the 20% piperidine in DMF solution, wash the resin thoroughly four times with 0.2 mL of DMF.

[0059] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-C(Bhoc)-OH (2.0 mg), oxyma (0.4 mg), and N,N'-diisopropylcarbodiimide (0.43 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF.

[0060] Add 0.2 mL of acetic anhydride blocking reagent (DMF: acetic anhydride: 2,6-lutidine = 89:5:6, volume ratio) and incubate the resin at room temperature for 2 minutes. After draining the acetic anhydride blocking reagent, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protecting group removal procedure. After draining the 20% piperidine in DMF, wash the resin four times with 0.2 mL of DMF.

[0061] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-T-OH (3.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0062] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-A(Bhoc)-OH (4.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0063] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-C(Bhoc)-OH (4.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0064] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-A(Bhoc)-OH (4.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0065] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-A(Bhoc)-OH (4.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0066] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-C(Bhoc)-OH (4.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0067] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-T-OH (3.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0068] Add 45 µL of NMP-based peptide nucleic acid condensation reagent containing Fmoc-PNA-A(Bhoc)-OH (4.0 mg), oxyma (0.8 mg), and N,N'-diisopropylcarbodiimide (0.86 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0069] Add 135 µL of DMF condensation reagent containing thioglycolic acid (2.0 µL), oxyma (4.0 mg), and N,N'-diisopropylcarbodiimide (4.4 µL). Incubate the resin at 55°C with shaking for 40 minutes. After draining the reaction mixture, wash the resin four times with 0.2 mL of DMF. Add 0.1 mL of 20% piperidine in DMF and incubate at room temperature for 6 minutes. Repeat the Fmoc protection group removal procedure and wash the resin four times with DMF.

[0070] The resin was transferred to a 1.5 mL centrifuge tube and incubated at room temperature for 80 minutes with 1.5 mL of dithiothreitol / N,N-diisopropylaminoethylamine / NMP / water (231 mg / 261 µL / 1.1 mL / 150 µL). The resin was then washed with DMF and then with DCM / DMF (1:1). 160 µL of a condensation reagent (1:1 DCM / DMF) containing Boc-Tyr(tBu)-OH (16.8 mg, 50 µmol), 4-dimethylaminopyridine (0.3 mg), and N,N'-diisopropylcarbodiimide (4.0 µL) was added. The resin was incubated at room temperature with shaking for 14 hours. The resin was washed sequentially with DMF and DCM and air-dried. 0.5 mL of fresh trifluoroacetic acid cleavage buffer (trifluoroacetic acid:phenol:water:triisopropylsilane = 88:5:5:2, v / v) was added.

[0071] After incubation at room temperature for 2 hours, the trifluoroacetic acid lysate was collected, 8 volumes of ice-cold ether were added, and the crude PNA3 was obtained by centrifugation. The crude PNA3 was dissolved in 0.1% water. The structural formula, HPLC analysis, and mass spectrometry analysis of PNA3 are shown in Figure 2. Figure 4 As shown, confirm the correctness of the product (molecular formula: C 107 H 133 N 53 O 28 The target PNA3 was purified by semi-preparative chromatography and freeze-dried to obtain a powdered PNA3 molecule (3.0 mg).

[0072] Example 4: According to the above content Acylation modification of RNA1 guided by donor template DNA1 Prepare four 1.5 mL RNase-free microcentrifuge tubes, designated ep1, ep2, ep3, and ep4. Add 5.0 µL of 200 mM imidazole-acetic acid buffer (pH 7.9), 1.0 µL of DNA1 (125.0 µM), and 1.0 µL of RNA1 (125.0 µM) to tubes ep1, ep2, and ep3. Add 5.0 µL of 200 mM imidazole-acetic acid buffer (RNase-free, pH 7.9), 1.0 µL of RNase-free H₂O, and 1.0 µL of RNA1 (125.0 µM) to tube ep4. Heat all four tubes at 95°C for 2 minutes, then incubate at room temperature for 15 minutes. Centrifuge each tube at room temperature for 30 seconds, then add 2.0 µL of RNase-free H₂O to each tube. To tubes ep1, ep2, and ep3, 1.0 µL of PNA1 (156.25 µM), 1.0 µL of PNA2 (156.25 µM), and 1.0 µL of PNA3 (156.25 µM) were added, respectively. To tube ep4, 1.0 µL of PNA1 (156.25 µM) was added. After incubating the four tubes at 12°C for 2 hours, 40.0 µL of 0.3 M NaOAc (pH 5.2) and 100.0 µL of absolute ethanol were added. The four tubes were centrifuged at 15,000 × g for 15 minutes at room temperature. After discarding the liquid from the four tubes, 8.0 µL of RNase-free H₂O (HO) was added to dissolve the precipitate. Subsequently, 1.0 µL of 10× Reaction Buffer (containing MgCl₂) and 1.0 µL of DNase I (1.0 U, Bio-Tech Catalog No. #B618252) were added. The tubes were incubated at 37°C for 15 minutes. 40.0 µL of 0.3 M NaOAc (pH 5.2) and 100.0 µL of absolute ethanol were added. The four tubes were centrifuged at 15,000 × g for 15 minutes at room temperature. Acylated RNA1 was generated in tubes ep1, ep2, and ep3.

[0073] After discarding the contents of the four centrifuge tubes, 8.0 µL of 500 mM HEPES-KOH buffer (RNase-free, pH 7.5), 1.0 µL of RNase-free H2O, and 1.0 µL of DMSO-biotin N-hydroxysuccinimide activated ester (7 nmol) were added. The tubes were incubated at 16°C for 60 minutes. 40.0 µL of 0.3 M NaOAc (pH 5.2) and 100.0 µL of absolute ethanol were added. The four tubes were centrifuged at 15,000 × g for 15 minutes at room temperature. After discarding the contents of the four tubes, 50.0 µL of 70% ethanol was added to each tube, and the mixture was centrifuged at 15,000 × g for 15 minutes at room temperature. The contents were then discarded. 8.0 µL of 10.0 mM NaOAc (pH 5.2) containing 5 µg of streptavidin protein was added to each tube. After incubating at 16°C for 25 minutes, add 24.0µL of acidPAGE loading buffer (1.0 mL consists of 50.0µL of 3.0 M NaOAc (pH 5.2), 20.0µL of 0.5 M EDTA (pH 8.0), 930µL of formamide, and 8.0µL of 2% (w / v) bromophenol blue). Load 20µL of the sample and run on an acid polyacrylamide gel (20%, 50.0 mM NaOAc (pH 5.2) running buffer, 120V, 100 minutes). Collect the gel and stain with ExRed (10000x). Observe the sample during gel electrophoresis, as shown in Figure 5. Figure 5 As shown (Lane 1 is pure RNA1; Lane 2 is the sample from centrifuge tube ep1; Lane 3 is the sample from centrifuge tube ep2; Lane 4 is the sample from centrifuge tube ep3; Lane 5 is the sample from centrifuge tube ep4; Lane 6 is the sample from pure DNA1). The figure shows that in the presence of donor PNA1 (or donor PNA2, or donor PNA3) and template DNA1, RNA1 produces distinct acylated product bands. In the absence of template DNA1, RNA1 produces no acylated products, demonstrating that acylation of RNA1 occurs under the guidance of template DNA1.

[0074] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for RNA acylation modification, characterized in that: The method for RNA acylation modification comprises the following steps: S1. The receptor RNA molecule and the template DNA molecule are hybridized after high-temperature denaturation and low-temperature annealing in a buffered salt solution to form a receptor RNA•template DNA hybrid; S2. Through the principle of complementary base pairing, the donor PNA molecule hybridizes with the above receptor RNA•template DNA hybrid to form a receptor RNA•template DNA•donor PNA double helix; S3. Through the adjacent space effect, the acyl group of the donor PNA molecule on the acceptor RNA, template DNA, and donor PNA double helix is ​​transferred to the hydroxyl group of the 3'-terminal ribose of the acceptor RNA to form a nucleic acid sample; S4. The nucleic acid sample is subjected to the action of deoxyribonuclease DNase I to degrade the template DNA, and then the target acylated RNA molecule is separated.

2. The method according to claim 1, wherein: The ribose at the 3' end of the receptor RNA molecule contains at least one hydroxyl group, and the receptor RNA molecule is any one of messenger RNA, ribosomal RNA, transfer RNA, small nuclear RNA, microRNA, small nucleolar RNA or non-coding RNA in a single-stranded RNA molecule.

3. The method according to claim 1, wherein: The N-terminus of the donor PNA molecule is connected to the acyl group to be transferred via a phenol ester or a thioester, and the donor PNA molecule has the following general formula: where X a For phenolic esters: , Aryl thioesters: or alkyl thioesters: Any one of the following; R is any one of a natural aminoacyl group, a non-natural aminoacyl group, an oligopeptide acyl group composed of natural amino acids, an oligopeptide acyl group containing non-natural amino acids, an acyl group containing a natural or non-natural monosaccharide, an acyl group containing a natural or non-natural polysaccharide, an acyl group containing a peptide nucleic acid or nucleic acid, an acyl group of a natural amino acid protein, an acyl group of a non-natural amino acid protein, or an acyl group containing a lipid; Z is any one of a hydrogen, an electron-donating, or an electron-withdrawing substituent on the benzene ring; X b X is any one of hydrogen, a side chain group of a natural amino acid except glycine and proline, or a side chain group of a non-natural amino acid; c Any one of the four bases of deoxyribonucleic acid; X d Any one of the four bases of deoxyribonucleic acid; X e It is any one of the oligopeptide sequences composed of amino groups, natural or non-natural amino acids; N is nitrogen; H is hydrogen; O is oxygen; S is a sulfur atom; n is any number between 0 and 5; and m is any number between 2 and 49.

4. The method according to claim 1, wherein: The template DNA molecule is designed to simultaneously hybridize with the acceptor RNA and the donor PNA to form a double helix, and the hydroxyl group of the ribose at the 3' end of the acceptor RNA is spatially close to the acylated group connected to the N-terminus of the donor PNA.

5. The method according to claim 1, wherein: The buffered saline solution in step S1 is any one of PBS, HEPES, NaOAc, Tris and imidazole, and has a pH range of 6.5-10.

0.

6. The method according to claim 1, characterized in that In the double helix structure of the acceptor RNA, template DNA, and donor PNA in step S2: there are no other bases between the bases paired between the 3' end of the acceptor RNA and the template DNA and the bases paired between the N end of the donor PNA and the template DNA, and the hydroxyl group of the ribose at the 3' end of the acceptor RNA is spatially close to the acyl group of the donor PNA; the number of paired bases in the region where the acceptor RNA and the template DNA undergo base complementary pairing is any number between 5 and 500; the number of paired bases in the region where the donor PNA and the template DNA undergo base complementary pairing is any number between 3 and 50.

7. The method according to claim 1, wherein: The concentration range of the acceptor RNA, template DNA and donor PNA in steps S1, S2 and S3 is 0.001 μM to 100.0 mM, and the reaction time is 5 min to 72 h, and the reaction temperature is 0 to 75°C.

8. The method according to claim 1, wherein: The method for isolating the target acylated RNA molecule in step S4 is any one of high performance liquid chromatography separation, size exclusion chromatography, ethanol precipitation, isopropanol precipitation, phenol-chloroform extraction, nucleic acid purification column, PAGE purification or Trizol extraction.

9. An application of the RNA acylation modification method according to any one of claims 1 to 8 in RNA interference therapy, antisense oligonucleotides, small activating RNA therapy, messenger RNA-based therapy, mRNA display of non-standard amino acid cyclic peptides, ribosome display, in vitro expression of protein macromolecular drugs, in vitro ribosomal translation of non-natural amino acids in cell-free systems, and in vitro genetic codon reprogramming.

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