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CN-122012495-A - 5' -End sequence rearranged RNA molecule and application thereof in detection of capping rate of co-transcribed capped target RNA

CN122012495ACN 122012495 ACN122012495 ACN 122012495ACN-122012495-A

Abstract

The invention discloses a5 'end sequence rearranged RNA molecule, which is provided with a 5' UTR, wherein the nucleotide sequence of the 1 st to 5 th positions of the 5 'end of the 5' UTR is GN 2 N 3 N 4 N 5 , N 2 is G or A, N 3 is G, A or T, N 4 is G, A or T, N 5 is G or T, at least 1 base in N 2 -N 5 is G, and the bases of N 3 -N 5 are not G at the same time. The invention also discloses a preparation method of the 5 '-end sequence rearranged RNA molecule and application of the 5' -end sequence rearranged RNA molecule in serving as an uncapped RNA standard. The invention also discloses a capping rate detection method of the target RNA and a kit for detecting the capping rate of the target RNA. The invention can prepare the 5' end sequence rearranged RNA molecule by in vitro transcription under the condition of not adding cap analogues, and can be used as an uncapped RNA standard substance for detecting the capping rate of co-transcribed capping production target RNA.

Inventors

  • WANG YUCAI
  • GUO YAKUN
  • WU YU
  • LIU WEIWEI
  • SHEN YANQIONG
  • HAN JING

Assignees

  • 合肥综合性国家科学中心大健康研究院

Dates

Publication Date
20260512
Application Date
20251230

Claims (10)

  1. 1. A5 ' end sequence rearranged RNA molecule is characterized in that the 5' end sequence rearranged RNA molecule is provided with a 5' UTR, the nucleotide sequence of the 5' end 1-5 position of the 5' UTR is GN 2 N 3 N 4 N 5 , N 2 is G or A, N 3 is G, A or T, N 4 is G, A or T, N 5 is G or T, and at least 1 base in N 2 -N 5 is G and the base in N 3 -N 5 is not G at the same time.
  2. 2. The 5 'end sequence rearranged RNA molecule of claim 1, wherein the nucleotide sequence at the 1 st to 5 th positions of the 5' end sequence rearranged RNA molecule 5'UTR is one of GATGG, GGATG, GGGAT, GAGTG, preferably the nucleotide sequence of the 5' end sequence rearranged RNA molecule 5'UTR is shown as SEQ ID NO. 1-4, preferably the nucleotide sequence of the 5' end sequence rearranged RNA molecule 5'UTR is shown as SEQ ID NO. 3, preferably the 5' end sequence rearranged RNA molecule is one of mRNA molecule and saRNA molecule.
  3. 3. A method for producing a 5' -end sequence-rearranged RNA molecule according to claim 1 or 2, characterized in that the 5' -end sequence-rearranged RNA molecule is obtained by subjecting a DNA sequence template of the 5' -end sequence-rearranged RNA molecule according to claim 1 or 2 to transcription reaction using an in vitro transcription system without adding a capping analog.
  4. 4. The method for preparing a 5' end sequence rearranged RNA molecule according to claim 3, comprising the steps of constructing a DNA sequence template of the 5' end sequence rearranged RNA molecule according to claim 1 or 2, and carrying out transcription reaction on the DNA sequence template by using an in vitro transcription system under the condition of not adding a capping analogue to obtain the 5' end sequence rearranged RNA molecule.
  5. 5. Use of a5 'end sequence rearranged RNA molecule according to claim 1 or 2, a 5' end sequence rearranged RNA molecule produced according to the method of claim 3 or 4, as an uncapped RNA standard, preferably for detecting the capping rate of a target RNA as an uncapped RNA standard, preferably for detecting the capping rate of co-transcribed capped production target RNA as an uncapped RNA standard.
  6. 6. The use according to claim 5, wherein the target RNA has a 5' cap structure, preferably the target RNA has a 5' UTR with a 5' end sequence rearranged RNA molecule, the length of the 5' UTR sequences are the same, the nucleotide sequence from position 6 at the 5' end of the 5' UTR sequences are the same, preferably the sequence of the target RNA is the same as the sequence of the other region of the 5' end sequence rearranged RNA molecule except for the 5' cap structure, the 5' UTR region.
  7. 7. The use according to claim 5 or 6, wherein the nucleotide sequence of the 5' UTR of the target RNA is shown in SEQ ID NO. 5.
  8. 8. The capping rate detection method of the target RNA is characterized by comprising the following steps of detecting a target RNA sample produced by co-transcription capping by adopting a high performance liquid chromatography, a mass spectrometry, a liquid chromatography-mass spectrometry or a capillary electrophoresis method, and qualitatively detecting the capping rate by using an uncapped RNA standard; wherein the uncapped RNA standard is a 5 'end sequence rearranged RNA molecule of claim 1 or 2 or a 5' end sequence rearranged RNA molecule produced according to the method of claim 3 or 4.
  9. 9. The method for detecting the cap ratio of a target RNA according to claim 8, wherein the target RNA has a 5 'cap structure, preferably the target RNA has a 5' UTR with the 5 'end sequence rearranged RNA molecule having the same length as the 5' UTR sequence, the 5 'ends of the 5' UTR sequences have the same nucleotide sequence from the 6 th position, preferably the target RNA has the same sequence as the other region of the 5 'end sequence rearranged RNA molecule except the 5' cap structure and the 5'UTR region, preferably the nucleotide sequence of the 5' UTR of the target RNA is shown as SEQ ID NO: 5.
  10. 10. A kit for detecting the capping rate of a target RNA, which is characterized by comprising an uncapped RNA standard, wherein the uncapped RNA standard is a 5 'end sequence rearranged RNA molecule according to claim 1 or 2 or a 5' end sequence rearranged RNA molecule prepared according to the method of claim 3 or 4, preferably the target RNA is a target RNA produced by co-transcription capping, preferably the target RNA has a 5 'cap structure, preferably the target RNA and the 5' end sequence rearranged RNA molecule both have 5'UTR, the 5' UTR sequences of which are the same in length, the 5 'ends of the 5' UTR sequences are the same in nucleotide sequence from the 6 th position, preferably the target RNA and the other regions of the 5 'end sequence rearranged RNA molecule are the same except the 5' cap structure and the 5'UTR region, and preferably the nucleotide sequence of the 5' UTR of the target RNA is as shown in SEQ ID NO: 5.

Description

5' -End sequence rearranged RNA molecule and application thereof in detection of capping rate of co-transcribed capped target RNA Technical Field The invention relates to the technical field of biological medicine, in particular to a 5' -end sequence rearranged RNA molecule and application thereof in detecting the capping rate of co-transcribed capped target RNA. Background The sequence of mRNA comprises, from the 5 'end to the 3' end, a 5 'non-coding sequence (UTR), a target protein coding sequence, a 3' non-coding sequence (UTR) and a polyA tail. Self-replicating RNA (saRNA) comprises, from the 5 'end to the 3' end, a 5 'non-coding sequence (UTR), an open reading frame encoding an RNA replicase, a subgenomic promoter (SGP), a protein coding sequence of interest, a 3' non-coding sequence (UTR), and a polyA tail. The target protein coding sequence is replaced by a virus structural protein coding region, and other sequences are all from a virus genome, which is the currently mainstream saRNA sequence design concept. since saRNA has the characteristics of low dosage, high expression, self-amplification and the like, saRNA has been widely paid attention to the fields of tumor vaccines, infectious disease vaccines and gene therapy in recent years. At present, co-transcription capping technology (co-transcriptional capping) is commonly adopted in industrialized mRNA and sarNA production. The process adds capping analogues (such as ARCA, cleanCap and the like) into an in-vitro transcription system, so that the RNA synchronously obtains a 5' end cap structure in the transcription process, thereby improving the stability and translation efficiency of the RNA, and being simple, convenient and efficient in process. In co-transcriptional capping systems, higher proportions of capped RNA products are typically obtained due to the influence of factors such as the template promoter, the 5' end sequence characteristics, and the involvement of the capping analog in transcription initiation. However, under the condition of not adding a capping analogue, high-yield and reproducible uncapped RNA can be obtained by using the same template, and partial sequences (such as target RNA sequences, the 5'UTR nucleotide sequence of which is shown as SEQ ID NO: 5) are limited by factors such as transcription initiation efficiency, 5' end structure, RNA stability and the like, so that the yield of uncapped RNA is extremely low and the batch-to-batch variation is large. These sequences have a great impact on transcription efficiency due to the presence or absence of cap analogues, which are presumably added to the 5' end in absolute advantage in the co-transcriptional capped IVT reaction. In preparing mRNA or sarNA using the co-transcriptional capping process, the capping rate of the final product needs to be monitored for quality monitoring. Common capping rate detection means include HPLC, LC-MS, capillary electrophoresis, and the like. At present, an LC-MS method is commonly adopted, when the capping rate is quantitatively detected by a normalization method, the molecular weights of capped RNA and uncapped RNA are generally determined according to the 5' -end shearing sequence of target RNA, the peak positions of the capped RNA and the uncapped RNA in an LC-MS map are determined according to the molecular weights, and then the relative capping rate is calculated by the normalization method. This detection method theoretically does not require the use of standards. However, in the actual detection, a part of the sample to be detected has a result that uncapped RNA is not detected, and the result can lead people to whether chromatographic conditions are proper or not and whether uncapped RNA can be detected or not to question, so that the accuracy of the detection result needs to be further determined by detecting an uncapped RNA standard. As described above, when uncapped RNA is prepared by an in vitro transcription method without adding a capping analog, the yield is extremely low, and it is difficult to prepare an uncapped RNA standard. The cost for obtaining the uncapped RNA standard substance by a chemical synthesis mode is high, and the natural base RNA is easy to degrade and has a plurality of impurities in the chemical synthesis and purification process, thereby being not beneficial to large-scale application and obtaining the standard substance which is highly consistent with the process of an actual product. When an uncapped RNA standard that is highly consistent in length and molecular weight with the target mRNA or target saRNA, but does not carry a cap structure, cannot be obtained, detection of the cap rate can only be performed using an indirect or relative quantitative method, which can greatly reduce the accuracy and comparability of cap rate assessment. Thus, there is a need in the art for an uncapped RNA standard that can be efficiently prepared by in vitro transcription while maintaining substantial agreement with th