CN108314736B - Method for promoting RNA degradation - Google Patents

Abstract

The invention discloses a method for promoting RNA degradation, which connects ribonuclease H with a DNA binding domain through a genetic engineering method, and when exogenous DNA with specific recognition characteristics of the DNA binding domain is bound on RNA, the endoribonuclease activity of the ribonuclease H of a fusion protein can degrade the RNA in an RNA/DNA hybridization system. The method realizes the specific and efficient degradation of the RNA of the nucleic acid molecule, can be used for inhibiting gene expression in vivo and clearing RNA virus, and is simple and practical.

Description

Method for promoting RNA degradation

Technical Field

The invention belongs to the technical field of genetic engineering, and relates to a method for promoting RNA degradation, in particular to a method for degrading RNA in an RNA/DNA hybrid system based on a ribonuclease H combined DNA binding domain.

Background

The target RNA is degraded, so that the method has a great application prospect:

1. application in exploring gene function: the post-genome era of elucidating the biological effects of functional gene expression products in the genome has profound significance for medical development. The RNA degradation technology can remove the target gene expression in a sequence specific mode quickly, economically and simply, and becomes an important research means for exploring gene functions. Has important significance for clarifying signal transduction paths and discovering new drug action targets.

Use of RNA degradation technology in the field of gene therapy: the RNA degradation technology is used as an efficient sequence-specific gene knockout technology to develop rapidly in the field of infectious diseases and malignant tumor gene therapy. In the gene therapy research of HIV-1, hepatitis B, hepatitis C and the like by utilizing RNAi technology, the fact that the sequence which is not homologous with the human genome in the viral genome is selected as an inhibition sequence can inhibit the replication of the virus and avoid toxic and side effects on normal tissues. Meanwhile, the inhibitory sequence is selected at a specific site, and can generate apoptosis induction effect on malignant tumor cells with definite gene mutation in part, such as leukemia cells containing BCL/ABL or AML1/MTG8 fusion genes. In addition, the tumor specific promoter such as hTERT promoter, survivin promoter or tissue specific promoter such as tyrosinase promoter and osteocalcin promoter can be used for guiding the expression of siRNA or shRNA aiming at certain oncogene or anti-apoptosis molecule, thereby achieving the purpose of specifically killing tumor cells.

3. Treatment of viral diseases: researchers at the university of california los angeles university and the california school of science and technology developed the use of RNAi technology to prevent hiv entry into human cells. These results suggest that RNA degradation technology is competent for gene therapy of many viruses and that RNA degradation technology will become an effective antiviral therapeutic approach. This is of great significance for the control of many serious infectious diseases in animals.

4. Treatment of genetic diseases: carthew R W at the northwest university in the United states and Ishizuka A et al, the Japanese institute for genetic research, found that RNAi is closely related to fragile X syndrome (a chromosomal disorder associated with FMR-1 gene abnormality that causes mental retardation), and revealed that defects in the mechanisms associated with RNAi may lead to pathological mechanisms of human diseases. RNA degradation therapy of genetic diseases is a major focus of research today.

Currently, RNA degradation technologies are mainly RNAi based on RNA interference and antisense oligonucleotide technology based on DNA. The degradation effect of these techniques has not always been able to achieve a high efficiency goal. The invention develops a novel RNA degradation technology. This technology is based on a method of degrading RNA in RNA/DNA hybrid systems by ribonuclease H in combination with a DNA binding domain. The technology introduces a DNA binding domain to enable ribonuclease H to find an RNA/DNA hybrid chain more quickly and accurately and degrade RNA.

Disclosure of Invention

The technical problem to be solved is as follows: the invention aims to overcome the defects of the existing RNA degradation technology and provides a method for degrading RNA, which has the advantages of high efficiency, simplicity, economy and accuracy.

The technical scheme is as follows: a method for promoting RNA degradation by using antisense oligonucleotide with DNA binding domain binding structure (such as 5' end phosphorylation modification and self-forming hairpin structure or another short phosphorylated oligonucleotide and antisense oligonucleotide forming double-stranded structure), and degrading RNA in RNA/DNA hybrid system under the action of fusion protein with characteristic DNA binding domain and ribonuclease H and corresponding characteristic DNA.

In order to improve the stability of the antisense oligonucleotide in cells, partial nucleotides in the antisense oligonucleotide can be modified by phosphorothioate.

The fusion protein with the characteristic DNA binding domain and the ribonuclease H can be obtained by prokaryotic expression (such as escherichia coli) and purification:

the expressed protein sequence is shown in SEQ ID NO.1, and in order to ensure that the protein can smoothly enter cells, a cell-penetrating peptide sequence (RRRRRRRRRR) is added at the end N; in order not to affect the respective functions of the DNA binding domain and ribonuclease H, a flexible peptide (GGGGSGGGGSGGGG) is added between them; meanwhile, for the convenience of purification, a histone purification tag (HHHHHHHH) was added to the C-terminus. In order to obtain the active protein more efficiently, the codon for translating the protein is comprehensively optimized by software so that the protein is more suitable for being expressed in escherichia coli, and the protein is constructed on a prokaryotic expression vector for expression and purification of the escherichia coli.

The fusion protein with the characteristic DNA binding domain and the ribonuclease H can also construct a eukaryotic expression vector, and is expressed by directly transfecting cells through viruses or plasmids:

the expressed protein sequence is shown in SEQ ID NO.2, and compared with SEQ ID NO.1, the protein has the N-terminal removed and is added with a membrane-penetrating peptide sequence and a histone purification tag sequence. In order to obtain active protein more efficiently, the codon for translating the protein is comprehensively optimized by using software so as to be suitable for expression in eukaryotic cells, and the codon is constructed on a eukaryotic expression vector or a virus vector for intracellular expression.

The method of the invention is carried out according to conventional molecular biology methods.

The method is applied to the expression of interference genes or the elimination of RNA viruses and the like.

Has the advantages that: because the DNA binding structure domain is added on the basis of the ribonuclease H, the method greatly improves the RNA capability of the ribonuclease H in degrading RNA/DNA hybrid system, compared with the traditional antisense oligonucleotide method, improves the expression efficiency of interfering genes and the RNA capability of degrading RNA, and is suitable for gene function research, RNA virus removal and other applications; the method is carried out according to the popular molecular biology method, and the required reagents and instruments are commonly used and do not need to be purchased specially.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

Example 1

A method for promoting RNA degradation comprises inhibiting 293T cell AMPK alpha gene expression. The method specifically comprises the following steps:

1) according to the sequence of SEQ ID NO.1, prokaryotic expression is carried out and purification is carried out to obtain fusion protein;

2) the antisense oligonucleotides of Table 1 were added to the purified protein. Then adding the mixture to the cultured cells;

TABLE 1 antisense oligonucleotide sequences

3) Culturing for 48 hr to inhibit AMPK alpha expression.

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Claims (3)

1. A method of promoting RNA degradation, comprising: degrading RNA in an RNA/DNA hybrid system under the action of a fusion protein with a characteristic DNA binding domain and ribonuclease H and corresponding characteristic DNA;

the sequence of the fusion protein is shown in SEQ ID NO.1,

the characteristic DNA is a DNA sequence containing a binding domain that binds to the RNA and the DNA binding domain.

2. The method for promoting degradation of RNA according to claim 1, wherein: the fusion protein with the characteristic DNA binding domain and the ribonuclease H is obtained by expression and purification of escherichia coli or eukaryotic cells.

3. The method for promoting degradation of RNA according to claim 1, wherein: the corresponding characteristic DNA is a DNA sequence containing the degraded target RNA, which is double-stranded DNA and is phosphorylated at the 5 terminal.