Method for marking RNA by site specificity

A site-specific, labeling technology, applied in the direction of fermentation, etc.

Pending Publication Date: 2021-11-05
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0006] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is a means for site-specific labeling of RNA, which realizes the flexible labeling of designated sites of RNA: the base type of the labelable site is not limited ; Can mark one or several sites in the single-base repeat fragment; the marked sites are not limited by the complex advanced structure, and can be located in single-stranded or double-stranded regions

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  • Method for marking RNA by site specificity
  • Method for marking RNA by site specificity
  • Method for marking RNA by site specificity

Examples

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Embodiment 1

[0040] In this example, accurate site labeling is carried out in the repeated base region of RNA, specifically, fluorescent labeling is performed on the middle site of the continuous UUU sequence in long-chain RNA, and the labeled product is detected and purified by fluorescent gel electrophoresis and HPLC . Design the RNA leader strand first, such as figure 1As shown, design and synthesize a DNA double-stranded template according to the sequence of the RNA guide strand and the target RNA, and then incubate the RNA guide strand, the DNA double-strand immobilized by the beads and RNA polymerase in the reaction tube, so that the three are within 30-37 At ℃, a stable "RNA polymerase-DNA template-RNA leader strand" ternary complex is formed, and the long-chain RNA is specifically labeled with the modified UTP site, and the fluorescently-labeled intermediate site of the continuous UUU sequence in the RNA is obtained. RNA. The specific process is as follows:

[0041] 1. Design th...

Embodiment 2

[0049] In this embodiment, the form of the RNA leader strand is more diverse, specifically: 1. The RNA leader strand has a complex structure. 2. The RNA leader strand has modifications. The RNA leader strand with a complex structure is hybridized to the DNA strand, so that the 3' end of the RNA forms complementary base pairing with the "vacuole" region of the DNA template.

[0050] In this example, the RNA leader strand itself is modified, and after being assisted by the DNA strand, the pairing efficiency of the RNA leader strand and the DNA template is improved, and RNA containing modification groups at multiple sites is synthesized. Such as Figure 4 As shown, the upper part of the figure shows that the solid-phase DNA template, RNA polymerase and RNA leader strand cannot form a transcription complex, because the RNA leader strand folds itself to form a complex structure, and its 3'-end forms intramolecular base pairing, resulting in The RNA leader strand cannot form a thre...

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Abstract

The invention discloses a method for marking RNA by site specificity, which relates to the field of RNA synthesis and marking and comprises the following steps: designing and preparing an RNA pilot chain; designing and preparing a DNA double-stranded template; carrying out site-specific labeling on RNA; and adding modified or common nucleotide (NTPs) into a solid-liquid mixed phase reaction system, and carrying out transcription extension at the 3'end of an RNA pilot chain to realize accurate site labeling. According to the invention, a solid-liquid transcriptional extension compound is formed to extend the 3 '-end of an RNA pilot chain, and site-specific labeling is carried out in the solid-liquid transcriptional extension process. The length of the RNA capable of being marked is not limited, the method can be used for marking short-chain and long-chain RNA, and the specific site of the RNA is flexibly marked: the base type of the site capable of being marked is not limited; one or more sites in a single-base repeated fragment can be marked; the marked site is not limited by a complex advanced structure and can be located in a single-chain or double-chain region.

Description

technical field [0001] The invention relates to the field of RNA synthesis and labeling, in particular to a method for site-specific labeling of RNA. Background technique [0002] RNA plays a vital role in information transmission and biological regulation in life activities. Site-specific marker RNA has a wide range of applications, such as real-time observation of RNA in living cells, increasing the stability of RNA, and improving the efficacy of RNA drugs and vaccines. effectiveness. [0003] Solid-phase chemical synthesis is a common method for synthesizing and labeling RNA, but the main limitation of this method is that the length of the synthesized RNA does not exceed 100 bases (nt). The PLOR (Position-selective labeling of RNA, site-specific labeling RNA) method reported by Nature in 2015 precisely controls the transcription process of RNA polymerase by limiting nucleoside triphosphates (NTPs), and realizes the position of the RNA chain during the transcription proce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34
CPCC12P19/34
Inventor 刘昱王思雨陈典
Owner SHANGHAI JIAO TONG UNIV
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