RNA (Ribonucleic Acid) 4-mercaptouracil specific labeling method and application thereof

A mercaptouracil, RNA4- technology, applied in the field of nucleic acid chemistry, can solve the problems of low reaction efficiency, harsh reaction conditions, oxidative damage of classical bases, etc., and achieve the effect of good selectivity and strong specificity

Active Publication Date: 2021-08-24
XI AN JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

4sU can be labeled with a biotin-pyridine (HPDP-biotin) linked by a disulfide bond. After enrichment, DTT breaks the disulfide bond to release the enriched 4sU RNA, but the reaction efficiency of this method is very low. Low
However, the processes of the above-mentioned methods are complicated, the reaction conditions are harsh, and the oxidants used will cause oxidative damage to the classical bases.

Method used

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  • RNA (Ribonucleic Acid) 4-mercaptouracil specific labeling method and application thereof
  • RNA (Ribonucleic Acid) 4-mercaptouracil specific labeling method and application thereof
  • RNA (Ribonucleic Acid) 4-mercaptouracil specific labeling method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0069] Example 1: Using the addition and elimination reaction of hydrazine hydrate to RNA 4sU according to the present invention, a structure similar to cytosine was obtained.

[0070] In vitro transcription of RNA with 4sU site, the 4 S U-RNA reacts with hydrazine hydrate, and the reaction conditions are: water as solvent, 10mM DTT buffer, heating at 50°C for 1.5h. After the reaction, the RNA was recovered by alcohol precipitation. The labeled RNA was reacted with rSAP and RNAse I at 37°C for 12 hours to degrade the nucleic acid into nucleosides. Finally, various nucleosides in the sample were detected by LC-MS / MS.

[0071] see figure 2 , for 4 S The ultraviolet absorption spectra before and after the reaction of U and hydrazine hydrate, it can be seen from this figure that hydrazine hydrate can convert 4 S U is completely reacted, and a new product cytidine analogue C* is obtained.

[0072] Nucleoside HPLC mass spectrometry after RNA degradation see image 3 , it can...

Embodiment 2

[0074] Example 2: Single-base recognition of 4sU using the addition and elimination reaction of hydrazine hydrate to 4sU according to the present invention

[0075] RNA with 4sU site was transcribed in vitro, and 4Su-RNA was reacted with hydrazine hydrate. The reaction conditions were water as solvent, 10mM DTTbuffer, and heating at 50°C for 1.5h. After the reaction, the RNA was recovered by alcohol precipitation. The labeled RNA was subjected to reverse transcription and PCR. PCR products were characterized by 15% PAGE and sequencing. A mutation from T to C occurred at the position of the original 4sU, therefore, this method realized the single base recognition of 4sU.

Embodiment 3

[0076] Example 3: Identification of Modification Positions of Naturally Occurring 4-Mercaptouracil

[0077] The total RNA was extracted from Escherichia coli, and the total RNA was reacted with hydrazine hydrate. The reaction conditions were water as a solvent, 10mM DTT buffer was added, and the reaction was carried out at 50°C for 1.5h. tRNA with specific primers of known sequence val Reverse transcription, PCR, and next-generation sequencing were used to identify and confirm the position of the naturally occurring modified 4-mercaptouridine.

[0078] For the process of subcloning in this example, see Figure 10 , for the results of next-generation sequencing of subcloned plaques, see Figure 11 , which shows the naturally occurring 4 S The U site is changed from the original A:T pairing to the C:T pairing, indicating that the method of the present invention can recognize naturally occurring 4 S U site.

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Abstract

The invention discloses an RNA (Ribonucleic Acid) 4-mercaptouridine specific labeling method and application thereof, and belongs to the technical field of nucleic acid chemistry. According to the method, a structure similar to cytosine (C) is generated under a mild condition by utilizing a reaction mechanism that hydrazine hydrate is used for carrying out addition elimination on RNA 4-mercaptouridine; in the reverse transcription process of RNA, the original A: T pairing at the position of 4sU is changed into C: G pairing, so that the position of 4sU is subjected to single base recognition. The method is combined with a sequencing technology and can be used for detecting the position of naturally existing 4sU and researching the dynamic state of a transcriptome.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid chemistry, and in particular relates to an RNA 4-mercaptouracil specific labeling method and application thereof. Background technique [0002] RNA is an important biomacromolecule in living organisms. In cells, RNA is in a dynamic system of continuous transcription, processing, and degradation. To understand the function of RNA, it is necessary to study the dynamics of its transcriptome. Sequencing technology can capture the transient information of RNA but cannot directly convey the dynamic information of transcriptome. Based on this, more and more labeling reagents, such as EU, BrU, 4sU and other nucleosides that are easy to be chemically modified, are transcribed into newly generated RNA through metabolism, and combined with sequencing technology, they can be used to study the dynamics of the transcriptome. Among these metabolic labeling methods, 4sU metabolic labeling is a widely used...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q1/6886C12Q2600/158C12Q2525/117C12Q2521/107C12Q2531/113C12Q2527/125Y02A50/30
Inventor 赵永席陈锋苏丽
Owner XI AN JIAOTONG UNIV
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