CN-122012672-A - Method for detecting mRNA capping rate

Abstract

The invention discloses a method for detecting mRNA capping rate, belonging to the field of biotechnology. The detection method comprises the steps of carrying out hybridization reaction on mRNA and a probe without base modification, carrying out enzyme digestion reaction on the product, adding magnetic particles into the reaction product, and carrying out washing and elution after incubation to obtain an mRNA pretreatment product. The invention detects the mRNA capping rate by using the non-modified probe, wherein the pretreatment flow of the mRNA is mainly improved, the higher detection fragment yield is obtained by optimizing the reaction condition of hybridization combination of the probe and the mRNA, the ratio of the probe in a capping rate detection sample can be obviously reduced by optimizing the elution mode, and the proportion of the target fragment is improved. Therefore, compared with the traditional detection method, the method can improve the detection sensitivity, reduce the detection lower limit, improve the resolution and the accuracy, simplify the flow, reduce the cost and have better application prospect.

Inventors

• ZHANG YIMIN
• CHEN WEIJIE
• WANG PENGYUN
• LIU BOTAO

Assignees

• 生工生物工程（上海）股份有限公司

Dates

Publication Date

20260512

Application Date

20260304

Claims (10)

1. 1. A method of detecting mRNA capping rate, the method comprising: The mRNA and the probe without base modification are hybridized, and the product is subjected to enzyme digestion reaction, then magnetic particles are added into the reaction product, and after incubation, the mRNA pretreatment product is obtained through washing and elution; The hybridization reaction is carried out under the conditions of heating at 68-72 ℃ for 2-10 min, ice bath for 0.5-1 h and incubation at 35-38 ℃ for 1-4 h.
2. 2. The method of claim 1, wherein the elution solution in the eluting step is nuclease free water.
3. 3. The method according to claim 2, wherein the elution method comprises adding nuclease-free water to the washed magnetic particles, heating at 60-80 ℃ for 2-5 min, and attracting the magnetic particles by a magnetic frame, thereby obtaining mRNA pretreatment product after treatment.
4. 4. The method of claim 1, wherein the hybridization reaction system comprises mRNA, abasic modified probes, and incubation buffers; The incubation buffer had a composition of 10mM Tris-HCl, pH=7.5, 0.1M LiCl.
5. 5. The method according to claim 4, wherein the molar ratio of mRNA to the probe without base modification in the reaction system is 0.8 to 1:1.
6. 6. The method according to claim 1, wherein the composition of the non-base modified probe is 5 '-4-6 nt RNA-4-6 nt DNA-10-20 nt RNA-biotin-3'.
7. 7. The method of claim 1, wherein the mRNA comprises a piu modified mRNA and an abasic modified mRNA.
8. 8. The method according to claim 1, wherein the reaction system of the cleavage reaction comprises hybridization reaction products, a reaction buffer and RNsae H enzymes, and the cleavage reaction is carried out at 37 ℃ for 0.5-1.5 h.
9. 9. The method according to claim 1, wherein the incubation after adding the magnetic particles is performed for 20-40 min at room temperature at 20-50 rpm on a rotary mixer.
10. 10. The method according to claim 1, wherein the washing is carried out by treating the incubated magnetic particles with a magnetic rack, removing supernatant, and washing with a magnetic bead washing buffer and a nuclease-free water sequentially.

Description

Method for detecting mRNA capping rate Technical Field The invention relates to the technical field of biology, in particular to a method for detecting mRNA capping rate. Background MRNA (messenger ribonucleic acid) is an important molecule in organisms, and its main function is to transfer genetic information in DNA and guide the synthesis of proteins. mRNA vaccines utilize this mechanism, which does not directly contain the virus, but rather contains mRNA encoding a specific portion of the virus, which when it enters human cells, the cells produce the virus as indicated by the mRNA, eliciting an immune response, and thus a body that is resistant. Compared with the traditional vaccine, the mRNA vaccine has flexible design and shorter production period, and as the design and development only need the gene of the virus, when the virus is mutated, a new vaccine can be obtained only by adjusting the sequence. mRNA vaccine brings mRNA technology to public view, and the mRNA technology not only can be used for vaccine production, but also has great potential in the aspects of treating genetic diseases, cancers, gene editing and the like. In vitro transcription (In Vitro Transcription, IVT) can use RNA polymerase to synthesize RNA in vitro. Most eukaryotic mRNAs have a 5 'cap structure that protects the 5' end of the mRNA from phosphatases and nucleases and facilitates translation initiation. RNA synthesized by in vitro transcription does not have a 5 'cap structure and requires an additional capping step to obtain a 5' cap structure. 2 types of capping, enzymatic capping and co-transcriptional capping, are commonly employed in mRNA process production. Enzymatic Capping utilizes RNA triphosphatase activity, guanylate transferase activity and guanine methyltransferase activity of vaccinia virus Capping enzyme to generate Cap0-mRNA, and then utilizes 2' -O-methyltransferase to generate Cap1-mRNA, and in the presence of Capping buffer (Capping buffer), guanylate Triphosphate (GTP), S-adenosylmethionine (SAM) and the like at a proper concentration, RNA can be capped within one hour, and correct orientation is ensured. Co-transcription capping is to add a certain proportion of cap analogues directly into an in vitro transcription reaction system, and the reaction can directly obtain CAPPED MRNA, which has been developed to the third generation of cap analogues. Whether enzymatic capping or co-transcription capping, the capping rate of the resulting mRNA is detected after the reaction is completed, and the capping rate is an important indicator reflecting the quality of the mRNA. MRNA is typically composed of hundreds to thousands of nucleotides, and there may be a difference of one nucleotide or methyl between capped mRNA and uncapped mRNA, which is difficult to capture by conventional separation analysis methods. The current common method is to obtain a 5' end mRNA fragment of tens of nt through enzymatic degradation or cleavage, and then distinguish and quantitatively detect a short fragment carrying a cap by utilizing an acrylamide gel electrophoresis, a capillary gel electrophoresis or an LC-MS system, thereby finally determining the mRNA capping rate. The most ideal scheme is to capture by a probe modified by a holoxymethyl group, and cut with RNase H to obtain fragments, which are detected by LC-MS. According to the scheme, the capturing efficiency is improved through the modification of the total oxymethyl group, higher recovery efficiency than other schemes can be obtained, and a better detection effect can be achieved by matching with the low detection limit of the LC-MS. However, the recovery efficiency of mRNA fragments in this protocol is still low, while the cost of the whole protocol is high due to the high price of the probe modified by the perfluoromethyl group. Meanwhile, the residual probe components in the mRNA fragment product of the scheme often reach more than 70%, and the content of the effective detection fragments is further extruded. In view of this, the present invention has been made. Disclosure of Invention The invention aims to provide a method for detecting mRNA capping rate, which can be used for obtaining more detection fragments, reducing the content of probes in a sample, improving the resolution and simultaneously reducing the cost. The invention is realized in the following way: The invention provides a method for detecting mRNA capping rate, which comprises the steps of carrying out hybridization reaction on mRNA and a probe without base modification, carrying out enzyme digestion reaction on the product, adding magnetic particles into the reaction product, and carrying out washing and elution after incubation to obtain an mRNA pretreatment product; the hybridization reaction conditions are that heating is carried out for 2-10 min at 68-72 ℃, ice bath is carried out for 0.5-1 h, and incubation is carried out for 1-4 h at 35-38 ℃. In some embodiments, the elution solution in the el