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Hammerhead ribozyme screened in vitro and capable of shearing RNA

A ribozyme, cell technology, applied in the field of RNA molecules, can solve the problem of insignificant effect and so on

Pending Publication Date: 2019-04-12
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sequence and structure of wild-type HHRz are compatible with intramolecular cleavage, which may be one of the important reasons why ribozyme tools designed based on wild-type HHRz are not effective in the treatment of HIV-1 and other diseases

Method used

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  • Hammerhead ribozyme screened in vitro and capable of shearing RNA
  • Hammerhead ribozyme screened in vitro and capable of shearing RNA
  • Hammerhead ribozyme screened in vitro and capable of shearing RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. In vivo screening of ribozymes that catalyze intermolecular cleavage based on toxin reporter genes

[0030] The naturally occurring hammerhead ribozyme ( figure 1 A) is a ribozyme that catalyzes intramolecular cleavage. There are two highly conserved catalytic active centers (large catalytic active center and small catalytic active center), which consist of three stem-loop structures (stem 1, protruding loop 1, stem 2, Protruding loop 2, stem 3, protruding loop 3) are connected, GUC is one of the ribozyme cleavage recognition sites, and the cleavage position is after GUC ( figure 1 A scissors shown). The efficient cleavage of this ribozyme in cells depends on the interaction between protruding loop 1 and protruding loop 2. When the ribozyme is cleaved at the protruding loop 3, an intermolecular cleavage ribozyme can be formed, but the interaction between the protruding loop 1 and the protruding loop 2 still needs to be retained-the cleavage efficiency of th...

Embodiment 2

[0046] Example 2. The ribozyme obtained by screening down-regulates the expression of red fluorescent protein and Spinach in Escherichia coli.

[0047] The ribozyme screened in vivo has two substrate-binding domains (the first substrate-binding domain and the second substrate-binding domain), located on both sides of the catalytic core domain, and the two substrate-binding domains are respectively linked to the preselected RNA The sequences of the substrates are complementary, while the core catalytic domain remains unchanged. In this example, red fluorescent protein RNA (SEQ ID NO 10) and a sequence of Spinach (SEQ ID NO 11) were selected as the target RNA, and the working ability of the preferred ribozyme obtained through screening in prokaryotic cells was investigated. The corresponding plasmid constructs are as figure 2 Shown in A, other conditions are with reference to embodiment 1.

[0048] The red fluorescent protein mCherry was constructed downstream of the green fl...

Embodiment 3

[0050] Example 3. The ribozyme obtained by screening down-regulates the expression of red fluorescent protein in human cancer cell line (Hela).

[0051] The preferred ribozymes obtained from the screening show better cutting efficiency for various genes in Escherichia coli, a prokaryotic cell. In this example, the ability of the ribozyme to down-regulate the red fluorescent protein (SEQ ID NO 10) expressed in a eukaryotic human cell line was tested. The ribozyme obtained by screening was constructed on the eukaryotic expression vector pmCherry by restriction endonucleases SalI and HindIII, and the recombinant ribozyme-containing vector was extracted with an endotoxin-free plasmid extraction kit to obtain a plasmid. 5 micrograms of the recombinant vector used 2000 (Liposome 2000) transfected Hela cells that were plated on a 6-well plate one day in advance. 24 hours after transfection, single cell suspension was obtained by trypsinization, and the red fluorescence intensity o...

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Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a novel in vitro screening method of ribozyme and ribozyme obtained through screening by the method and capable of catalyzing RNA shearing activity. According to the in vitro screening method, toxoprotein ibsC is used as a report gene, ribozyme obtained through in vitro transcription controls the expression of toxicity protein on the shearing effect of mRNA corresponding to the ibsC so as to influence cell survival, and therefore, active ribozyme is obtained through screening. According to the method, a complicated in vitro screening step is not needed, so that influence of an inactive RNA sequence on the screening success rate can be reduced to the largest extent, and a simple efficient common intra-cellular screening platform is provided for ribozyme evolution. The effect manner of the ribozyme obtained through screening with a substrate is mainly an intermolecular shearing manner, and is suitable for catalysis and shearing of site specificity of target substrate molecules outside cells, in prokaryotic cells and in eukaryotic cells, and expression reduction on selected genes is realized.

Description

technical field [0001] The invention relates to an RNA molecule with catalytic RNA splicing activity obtained through in vivo screening. Background technique [0002] Ribozyme is a single-stranded nucleic acid macromolecule (Ribozyme) with catalytic function. According to their molecular weight, ribozymes can be divided into macromolecular ribozymes and small molecular ribozymes. Small ribozymes include Hammerhead Ribozymes (HHRz), Varkud satellite ribozymes, hepatitis D virus ribozymes, hairpin ribozymes, coiled ribozymes, pistol ribozymes, hatchet ribozymes, etc. , ranging in size from 30 to 150 nucleotides, catalyzes itself to undergo intramolecular cleavage reactions at specific locations. Among them, ribozymes that catalyze intramolecular cleavage reactions, such as hammerhead ribozymes, hairpin ribozymes, and hepatitis D virus ribozymes, can be transformed into ribozymes that catalyze intermolecular reactions. Ribozymes may play an important role in the treatment an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113
CPCC12N15/113C12N2310/121
Inventor 唐卓黄鑫董娟
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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