CN-121975915-A - Method for detecting false uracil based on rolling circle amplification mononucleotide resolution and application

CN121975915ACN 121975915 ACN121975915 ACN 121975915ACN-121975915-A

Abstract

The invention provides a method for detecting false uracil based on rolling circle amplification mononucleotide resolution and application thereof, belonging to the technical field of nucleic acid chemical modification detection. The method comprises three steps, bisulfite treatment, ligase mediated lock-probe circularization and RCA. By converting the bisulfite derived ψ signal into a lock probe ligation gate, single nucleotide resolution detection can be achieved without qPCR, and RNA input is low. The method can qualitatively identify the psi in rRNA and mt-tRNA, and can absolutely quantify target RNA and simultaneously determine the stoichiometric ratio of site-specific psi. In peripheral blood, the method can distinguish samples of AML patients and healthy controls, supports site-specific ψ measurement as a candidate biomarker, combines GLORI-mediated deamination, and can be applicable to other RNA modification. The method has the potential to promote functional research and clinical transformation research.

Inventors

WANG YAFEN
WENG XIAOCHENG
ZHOU XIANG
LIU XIUMIN

Assignees

武汉大学

Dates

Publication Date: 20260505
Application Date: 20260127

Claims (10)

1. A method for detecting false uracil based on rolling circle amplification mononucleotide resolution, comprising: bisulphite treatment is carried out on the RNA sample to be detected, so that pseudouracil in the RNA sample is subjected to chemical conversion to obtain a psi-BS adduct; performing reverse transcription on the obtained psi-BS adduct to obtain cDNA which forms a preset gap at a position corresponding to the target pseudouracil site; hybridizing the obtained cDNA with a lock probe designed for a target pseudouracil locus, and carrying out a ligation reaction under the action of ligase; performing rolling circle amplification on the ligation reaction product to obtain an amplification product comprising repeating units; and detecting signals of the amplified products, so as to realize detection of pseudo uracil loci in the RNA sample to be detected.
2. The method of claim 1, wherein the predetermined gap is a single base or oligonucleotide deletion structure.
3. The method of claim 2, wherein the lock-in probe comprises a first flanking sequence and a second flanking sequence that are complementary paired with the cDNA sequence flanking the predetermined gap, respectively, and the length or sequence composition of the first flanking sequence and the second flanking sequence is used to modulate the detection sensitivity.
4. The method of claim 1, wherein the method detects the pseudouracil at a single nucleotide resolution.
5. The method of claim 1, wherein the signal detection comprises any one of fluorescence detection, electrochemical detection, or imaging detection.
6. The method of claim 1, wherein the test RNA sample is derived from total RNA, mRNA, tRNA, rRNA or IncRNA cells.
7. The method of any one of claims 1-6, wherein the rolling circle amplification uses DNA polymerase to isothermally amplify the ligated lock-in probes.
8. Use of the method for detecting pseudouracil according to any of claims 1-6, characterized by comprising the use of any of the following A1) -A3): A1 Detecting a specific RNA ψ modification site associated with a disease in a clinical sample as a biomarker for diagnosis or prognosis of the disease; A2 In biological studies, functional studies and quantitative analyses of specific ψ modification sites in cellular or tissue RNAs; a3 Coupled with GLORI chemical treatments for single nucleotide resolution N6-methyladenosine detection.
9. A kit for detecting pseudouracil in RNA, said kit comprising at least: A bisulfite treatment reagent for inducing a structurally identifiable reverse transcription abnormality at a pseudo uracil corresponding site; a reverse transcription reagent for generating cDNA forming a predetermined gap at the position corresponding to the pseudo uracil; A lock-in probe comprising flanking sequences complementary to sequences flanking the cDNA of the predetermined gap, respectively; A ligase reagent for catalyzing the closed ligation of the lock-probe when the lock-probe hybridizes to the predetermined nick cDNA; And the rolling circle amplification reagent is used for conducting rolling circle amplification on the connected locking type probes so as to generate a signal amplification product signal detection reagent corresponding to the pseudo uracil locus, and is used for realizing visual detection of the rolling circle amplification product.
10. The kit according to claim 9, for detecting RNA modifications when combined with a treatment step for other RNA modifications capable of generating structural abnormalities during reverse transcription after specific chemical pretreatment.

Description

Method for detecting false uracil based on rolling circle amplification mononucleotide resolution and application Technical Field The invention relates to the technical field of nucleic acid chemical modification detection, in particular to a method for detecting pseudouracil based on rolling circle amplification mononucleotide resolution and application thereof. Background Pseudouridine (ψ) is one of the most abundant post-transcriptional modifications in RNA, called "fifth ribonucleotide", widely present in rRNA, tRNA, snRNA and mRNA, and plays a key role in regulating RNA stability, structure and function, etc. Abnormal levels of ψ modification are associated with a variety of diseases (e.g., leukemia, mitochondrial myopathy, neurodevelopmental disorders, etc.). In recent years, the successful application of N1-methyl pseudouridine (m 1 ψ) in mRNA vaccine further highlights the importance of accurately detecting the modification of ψ in basic research and clinical application. At present, the detection method of the psi is mainly divided into two major categories, namely 1) a full transcriptome analysis technology (such as Pseudo-seq, BID-seq, PRAISE and the like) based on high-throughput sequencing, which can provide a global map but has high cost and complex flow, and is not suitable for rapid detection of a few specific sites, and 2) a detection technology (such as SELECT, BIHIND and the like) based on site specificity, which is dependent on quantitative PCR (qPCR), requires precise instruments and is difficult to realize visual visualization or gel electrophoresis reading results. In addition, in the prior art, detection of the pseudo uracil is focused on chemical differentiation or differential identification on a sequence comparison level, a general technical scheme capable of directly converting a molecular structure difference induced by the pseudo uracil into a triggerable detection reaction is not available, and particularly, single nucleotide resolution detection of a pseudo uracil locus in RNA is realized without high-throughput sequencing and real-time quantitative PCR, and a large technical challenge is still faced. Therefore, there is a need in the art to develop a single nucleotide resolution ψ detection method with high sensitivity, high specificity, simple operation, low cost and no need of depending on qPCR or complex instruments, so as to meet the requirements of rapid, accurate quantification and visual analysis of specific RNA modification sites in clinical diagnosis, basic research and drug development. Disclosure of Invention The invention aims at overcoming the defects of the prior art and providing a method for detecting false uracil based on rolling circle amplification mononucleotide resolution and application thereof, wherein the method utilizes the specific chemical marking of bisulphite to ψ, by combining lock-type probe design and rolling circle amplification signal amplification, single base resolution qualitative detection, quantitative analysis and naked eye visualization of specific psi loci in RNA samples are realized. In order to achieve the above purpose, the present invention adopts the following technical scheme: in a first aspect, the present invention provides a method for detecting pseudouracil based on rolling circle amplification single nucleotide resolution, comprising: bisulphite treatment is carried out on the RNA sample to be detected, so that pseudouracil in the RNA sample is subjected to chemical conversion to obtain a psi-BS adduct; performing reverse transcription on the obtained psi-BS adduct to obtain cDNA which forms a preset gap at a position corresponding to the target pseudouracil site; hybridizing the obtained cDNA with a lock probe designed for a target pseudouracil locus, and carrying out a ligation reaction under the action of ligase; performing rolling circle amplification on the ligation reaction product to obtain an amplification product comprising repeating units; and detecting signals of the amplified products, so as to realize detection of pseudo uracil loci in the RNA sample to be detected. Further, the predetermined gap is a single base or oligonucleotide deletion structure. Further, the lock-type probe comprises a first flanking sequence and a second flanking sequence which are respectively complementarily paired with cDNA sequences at two sides of the preset notch, and the lengths or the sequence compositions of the first flanking sequence and the second flanking sequence are used for adjusting the detection sensitivity. Further, the method detects the pseudouracil at a single nucleotide resolution. Further, the signal detection includes any one of fluorescence detection, electrochemical detection, or imaging detection. Further, the test RNA sample is derived from cell total RNA, mRNA, tRNA, rRNA or IncRNA. Further, the rolling circle amplification uses DNA polymerase to carry out isothermal amplification on the connected locking t