A Differential Nuclease Digestion Method and Its Application in Detection of Internal Modifications of mRNA by LC-MS Method

A nuclease and differential technology, applied in the field of differential nuclease cleavage combined with LC-MS, can solve the problem of separation and achieve the effect of simple operation

Inactive Publication Date: 2020-04-10
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, none of these methods can bind the m at the 5' end of the mRNA 7 G and m inside 7 G is distinguished, so to explore whether there is m in the mRNA 7 G needs to develop a new type that can distinguish the 5' end from the internal m 7 A highly selective, sensitive and accurate detection method for G

Method used

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  • A Differential Nuclease Digestion Method and Its Application in Detection of Internal Modifications of mRNA by LC-MS Method
  • A Differential Nuclease Digestion Method and Its Application in Detection of Internal Modifications of mRNA by LC-MS Method
  • A Differential Nuclease Digestion Method and Its Application in Detection of Internal Modifications of mRNA by LC-MS Method

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Experimental program
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Embodiment 1

[0034] Embodiment 1: S1 nuclease enzymatic hydrolysis, phosphodiesterase I enzymatic hydrolysis to the enzymatic cleavage efficiency of the 5' end cap structure of mRNA

[0035] Use mMESSAGE The T7Kit kit uses double-stranded DNA with a T7 promoter as a template for carrying 5' end m 7 In vitro transcription of RNA with G-cap structure, the 5' end of the RNA is the same as eukaryotic mRNA, and each RNA transcribed contains only one m at the 5' end 7 g. Use S1 nuclease and phosphodiesterase I to enzymolyze the RNA into nucleotides, then use alkaline phosphatase to enzymolyze the nucleotides into nucleosides, remove the protein, freeze and spin dry, and redissolve in 65 μL of water. Inject 50 μL and analyze by LC-MS. The enzymatic efficiency of S1 nuclease enzymatic hydrolysis and phosphodiesterase I enzymatic hydrolysis on the cap structure of the 5' end of mRNA can be calculated. See the results in image 3 , S1 nuclease and phosphodiesterase I have a huge difference in t...

Embodiment 2

[0036] Embodiment 2: Enzymolysis efficiency comparison of S1 nuclease enzymolysis, phosphodiesterase I enzymolysis and other enzymolysis methods

[0037] Use S1 nuclease, phosphodiesterase I, conventional enzymatic digestion (S1 nuclease and phosphodiesterase I share), exonuclease T, exonuclease I and T4 DNA polymerase to digest the same amount of mRNA For nucleotides, use alkaline phosphatase to enzymatically hydrolyze nucleotides into nucleosides, remove protein, freeze and spin dry, redissolve in 65 μL water, inject 50 μL, and analyze by LC-MS. Compare the peak areas of normal nucleosides (A, G, C, U) obtained by different enzymatic hydrolysis methods, the results are shown in Figure 4 .

Embodiment 3

[0038] Example 3: m 7 G standard and internal m of mRNA in rice samples 7 Chromatographic retention time comparison of G

[0039] Total RNA was extracted from rice samples using TRIzol reagent and reused The mRNA IsolationSystem kit is used to isolate and purify mRNA from rice total RNA. After the mRNA was hydrolyzed into nucleotides with S1 nuclease and phosphodiesterase I, the nucleotides were hydrolyzed into nucleosides with alkaline phosphatase, the proteins were removed, freeze-dried, and redissolved in 65 μL of water. Analysis of m by LC-MS 7 G standard, S1 nuclease digest and phosphodiesterase I digest. contrast m 7 Extracted ion chromatograms of G standard, S1 nuclease hydrolyzate and phosphodiesterase I hydrolyzate, the results are shown in Figure 5 .

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Abstract

The invention discloses a differential nuclease digestion method and application thereof to detecting mRNA internal modification through LC-MS. The differential nuclease enzyme-digestion method comprises, according to the characteristic of difference of S1 nuclease and phosphodiesterase I in digestion of the 5'-end cap structure of mRNA, digesting the mRNA into nucleosides through the S1 nuclease and the phosphodiesterase I, then performing LC-MS detecting analysis, and combining the detecting results of digestion through the two nucleases to differentiate N7-methyguanosine (m7G) in the cap and the inside of the mRNA and according to the achieve qualitative and quantitative analysis on the m7G inside the mRNA. The differential nuclease enzyme-digestion method is high in sensitivity and selectivity, can quantitatively detect the content of m7G inside encaryotic mRNA and further can be applied to study of related functions of the m7G inside the mRNA.

Description

technical field [0001] The invention relates to a method of differential nuclease cutting combined with LC-MS and its application in the analysis of modified nucleosides on mRNA. Background technique [0002] RNA modification refers to the modification on RNA bases A\T\C\G\U and ribose. It has been found that there are more than hundreds of nucleoside modifications and thousands of modification sites in RNA, most of which exist in rRNA and tRNA and affect the formation, structure and function of RNA. Recent studies have found that there are nucleoside modifications on mRNA, and these modifications are dynamic changes, which affect the expression of genes, which is another new way for organisms to regulate gene expression besides DNA modification. N 7 -Methylguanosine (m 7 G) is a special modification on eukaryotic mRNA because it constitutes a special cap structure at the 5' end of eukaryotic mRNA. the m 7 The cap structure of G is necessary for the growth of eukaryotic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/38C12Q1/686
CPCC12P19/38C12Q1/686C12Q2521/327C12Q2521/301C12Q2565/627Y02P20/582
Inventor 袁必锋冯钰锜褚洁梅
Owner WUHAN UNIV
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