RNAi,RNA干扰 z-bo 2020-09-17 413

　The phenomenon of RNA interference (RNAi) is an evolutionarily conserved defense mechanism against transgenic or foreign viruses. After introducing double strand RNA (dsRNA) with homologous complementary sequences to the target gene transcription product mRNA, it can specifically degrade the mRNA, resulting in the corresponding functional phenotypic deletion. This process belongs to transcription The category of posttracriptional gene siliencing (PTGS). RNAi is widespread in the biological world, from lower prokaryotes, to plants, fungi, invertebrates, and even recently discovered this phenomenon in mammals, but the mechanism is more complicated.

  One. Discovery of RNAi

As early as 1990, when conducting research on transgenic plants, it was accidentally discovered that some endogenous genes could not be expressed after the full-length or partial genes were introduced into plant cells, but the transcription of these genes did not have any effect, and this phenomenon was called genes. Posttracriptional gene silencing (PTGS). A similar phenomenon was found in Neuroora in 1996, but this phenomenon was named quelling of gene expression. It was first discovered that dsRNA can cause The clue to gene silencing comes from the study of the nematode Caenorhabditis elega. In 1995, Cornell University researchers Guo and Kemphues tried to use antisense RNA to block the expression of the par-1 gene in order to explore the function of the gene. As a result, the antisense RNA could indeed block the expression of the par21 gene, but strangely, Injecting sense strand RNA as a control also blocked gene expression. This strange phenomenon was not solved until 3 years later-Andrew Fire of the Carnegie Research Institute in Washington and Craig Mello of the University of Massachusetts Cancer Center first injected double-stranded dsRNA-a mixture of sense and antisense strands into nematodes, which induced Gene silencing is much stronger than injection of sense strand or antisense strand alone. In fact, only a few molecules of double-stranded RNA per cell are enough to completely block the expression of homologous genes. Later experiments showed that the injection of double-stranded RNA into the nematode can not only block the homologous gene expression of the entire nematode, but also cause the homologous gene silencing of its first generation offspring. They call this phenomenon RNA interference.

In the past, it was believed that there was no RNAi phenomenon in mammalian cells, because longer dsRNA can induce IFN (interferon) production in mammalian cells, and activate the transcription of PKR (dsRNA-dependent kinase) involved in the STAT pathway, and dsRNA itself Binding with PKR can also activate it, and then phosphorylate the translation initiation factor eIF2a to inactivate it, leading to non-specific protein synthesis obstacles; on the other hand, dsRNA can induce cells to produce a variety of antiviral proteins 2′,5 'Adenosine synthetase, generates 2',5' adenylate, activates non-specific RNase L, and produces non-specific RNA degradation effects. It has now been discovered that as long as dsRNA is shorter than 30, it will not promote the interferon effect, and at the same time it can specifically degrade mRNA and cause gene silencing, indicating that dsRNA can also play a role in mammalian cells, which is useful for future gene therapy, etc. RNAi applications provide new research directions.

　two. Mechanism of RNAi

In recent years, studies have found that small interfering RNA (or short interfering RNAs, siRNA) is an important intermediate effect molecule for RNA interference (RNAi). siRNA is a class of about 21-25 nucleotides in length. (nt) special double-stranded RNA (dsRNA) molecule, with a characteristic structure, that is, the sequence of the siRNA has homology with the target mRNA sequence; the two single-stranded ends of the siRNA are 5′ phosphate and 3′ hydroxyl In addition, there are 2 to 3 protruding unpaired bases at the 3'end of each single strand. The main process of RNAi is that dsRNA is cleaved by nuclease into 21-25 nt interfering small RNA (siRNA). siRNA mediates the recognition and targeted cleavage of homologous target mRNA molecules. The formation of dsRNA in cells is the first step of RNAi. dsRNA in cells can be formed through a variety of ways, such as the transcription product of DNA inverted repeats in the genome; Transcription of antisense and sense RNA; viral RNA replication intermediates; and the use of single-stranded RNA in cells as a template to synthesize dsRNA by cell or viral RNA dependent RNA polymerase (RdRp), etc. C. elega can be injected directly dsRNA or the nematode soaked in a solution containing dsRNA to introduce exogenous dsRNA, you can also get dsRNA by feeding bacteria that express sense and antisense RNA.

The mechanism of RNAi has been preliminarily clarified. The first step of RNAi is that dsRNA is processed and cleaved under the action of endonuclease (a nuclease with RNase Ⅲ-like activity) to form 21-25 nt sense and antisense sequences The small interfering dsRNA is composed of siRNA. The RNase III-like nuclease in Drosophila is called Dicer. Dicer contains helicase activity, dsRNA binding domain and PAZ domain. It has been found in Arabidois, C. elega, Schizosaccharomyces Dicer congeners also exist in pombe and mammals. The second step of RNAi is the formation of a ribosome by the antisense strand in the siRNA. This ribosome is called the RNA induced silencing complex (RNA induced silencing complex). , RISC), RISC-mediated cleavage of the region complementary to the siRNA antisense strand in the target mRNA molecule, so as to interfere with gene expression. RISC is composed of a variety of protein components, including endonuclease, exonuclease, and unwinding Enzymes and homologous RNA chain search activity, etc. The MUT7 (an RNase D-like protein) in C. elega may be a 3′5′ exonuclease. In addition, members of the PAZiwi protein family may be components of RISC Components, such as AGO1 in Arabidposis; QDE in N.Craa; RDE1 in C. elega, etc. These proteins are homologous to the translation factor eIF2C.

The latest research further reveals that ATP plays an important role in siRNA-mediated RNAi. The conversion of longer dsRNA to siRNA requires the participation of ATP. siRNA and protein factors form an inactive protein PRNA complex of about 360 kD; then The siRNA double-stranded structure unwinds and forms an active protein PRNA complex (RISC). This step is ATP-dependent. The RISC active complex recognizes and cleaves the target mRNA. This step may not require ATP to participate.

In addition, ATP brings phosphate molecules to the 5′ end of siRNA and plays an important role in the function of siRNA. The unwinding of the siRNA duplex structure in the above process is likely to be a stable structural change, because the siRNA duplex and cell lysate After incubation, ATP and other cofactors are removed, and the active complex containing siRNA can still recognize and cleave the target mRNA molecule.

iRNA can be used as a special primer to synthesize dsRNA with target mRNA as a template under the action of RNA-dependent RNA polymerase (RdRp), which can be degraded to form new siRNA; the newly generated siRNA can enter the above-mentioned cycle. This process It is called random degradative polymerase chain reaction (random degradative PCR). The nascent dsRNA is repeatedly synthesized and degraded to continuously produce new siRNAs, thereby gradually reducing the target mRNA and presenting gene silencing. RdRp generally only targets the expressed target mRNA This kind of specific amplification of target mRNA in the RNAi process helps to enhance the specific gene monitoring function of RNAi. Each cell only needs a small amount of dsRNA to completely shut down the corresponding gene expression, which shows that the RNAi process is biological The basic kinetic characteristics of catalytic reactions.

　　The difference between miRNA and siRNA:

　　 miRNA is single-stranded, while siRNA is double-stranded;

　　 miRNA participates in the regulation of growth and development genes under normal conditions, while siRNA does not participate in the normal growth of animals. siRNA can only be produced under the induction of viruses or other dsRNAs as a complement and supplement to miRNA;

　　 miRNA regulates gene expression at the post-transcriptional level, presumably it also works at the translation level, while siRNA regulates gene expression at the post-transcriptional level;

Dicer enzyme processes the two types of RNA. The miRNA is asymmetric, and only comes from the side arm of the RNA precursor containing the stem-loop structure. The remaining part is quickly degraded, and this asymmetry does not exist in the siRNA processing process. in.

　　三, the application prospects of RNAi

　　 Related issues in RNAi technology mainly involve the following points:

　　(1) Selection of dsRNA sequence dsRNA is mainly selected from the gene region in the open reading frame (ORF) of known cDNA. In order to prevent mRNA regulatory proteins from interfering with the binding of RISC and target RNA, selections should be avoided including: 1) the region downstream of the start codon or within the 50-100 nucleotide position of the stop codon; 2) the 5′ or 3′ end Translation region; 3) Intron region. In addition, polyguanylic acid sequence regions (≥3) should be avoided during sequence selection, because it is easy to form a tetramer structure and inhibit RNAi. Choose a fragment with a length of 21-23 nucleotides that is complementary to the sequence on the target mRNA. It is better to start with AA, because this method can simplify the dsRNA synthesis process and reduce the cost, and the synthesized dsRNA can better resist the degradation of RNase . In addition, try to make the GC content in the dsRNA sequence close to 50% (45%-55% is the best). High GC content can significantly reduce the effect of gene silencing. Before selecting, you can search the BLAST database to ensure that no other genes homologous to the target gene exist, and avoid causing silencing of other similar genes. Not all mRNAs are sensitive to RNAi. In order to ensure effective inhibition of target gene expression, it is best to synthesize two or more dsRNAs targeting different target regions of the same gene at the same time; moreover, marking the 3′ end of the dsRNA sense strand has no effect on the RNAi phenomenon, and the existing experiments have not yet It was found that the secondary structure of mRNA has any significant effect on RNAi. At present, RNAi technology mainly uses mammalian cells as the object to study the functions of their genes. However, dsRNA> 30 can cause interferon-like effects in mammalian cells, leading to non-specific reactions. Therefore, the downstream product molecule siRNA is directly used instead to achieve the purpose of gene research.

　　(2) Different organisms can choose different methods for dsRNA introduction. For simple organisms, such as single-cell organisms, electroporation can be used; for more complex organisms, dsRNA can be microinjected into germ cells or early embryos, and nematodes can also be injected into the intestine or prosthetic cavity, compared with microinjection , There is no significant difference in RNAi efficiency. There are soaking method, engineered bacteria feeding method, calcium phosphate co-precipitation method and so on. If the siRNA (sense strand and antisense strand) artificially synthesized by chemical method is used, it will be introduced into the target cell in the form of double strand after annealing process. Some people have proposed using plasmids or viruses as vectors, through transduction or transfection, using DNA as a template in the cell, using RNA polymerase III, and transcribing into siRNA (directly form a double strand or form a hairpin after being folded by palindrome sequence Structure), it can also produce more obvious RNAi effects. Recently, another new method of introduction is to use the external force of a high-pressure water gun to inject the constructed plasmid directly from the tail vein of a mouse to observe the gene silencing effect of RNAi on living biological models instead of cultured cells. The half-life of siRNA is short, and the process is more complicated due to the use of high-pressure external force, which limits its application in the treatment of human diseases. Pachuk et al. proposed an intramuscular injection method to introduce a plasmid expression vector targeting IL-12 siRNA into mice, and the RNAi effect obtained was longer.

　　(3) The hairpin-like structure of siRNA experiments proved that hairpin-like siRNA can prolong the action time in the cell. Such a structure can be formed by a chain of nucleotides having a palindrome sequence. But usually the palindrome structure is not easy to obtain, and it can be replaced by a symmetrical sequence of head-to-head meetings. The template for transcription of hairpin-like siRNA must be closely connected to the vector transcription promoter, and have the shortest polyadenylic acid tail as possible, so as to induce efficient RNAi effects. Paddison et al. proposed that a similar structure can also be applied to long dsRNA (around 500) without causing non-specific degradation of RNA. This provides a new way to detect gene function after long-term development in mammalian cells.

　　 RNAi application fields and prospects: RNAi is a highly efficient and specific gene blocking technology. It has developed rapidly in recent years and soon becomes a powerful tool for functional genomics research. Through experimental means, dsRNA molecules are introduced into cells to specifically degrade mRNAs that are homologous to their sequences in the cells, block endogenous gene expression, and study the functions of unknown genes in human or other biological genomes from the perspective of reverse inheritance. In the early days, this technology was used to isolate various components in the insulin signal transduction pathway of Drosophila. Recently, there have also been experimental reports on the various pathways involved in the process of studying intracellular lipid balance through RNAi. Prior to this, the complementary characteristics of antisense RNA and target mRNA sequence have been used to inhibit the occurrence of its phenotype. However, due to the weak inhibitory effect of antisense RNA on endogenously expressed genes, some transitional phenotypes are often produced. It causes errors in the judgment of gene function. Currently, the only drug that has been approved and considered to be clinically therapeutic is Vitravene. Compared with RNAi technology, it has higher specificity, faster action and less side effects. While effectively silencing target genes, it has no effect on the cell's own regulatory system. Recently, nearly 20 gene functions have been successfully "knocked out" in human somatic cells, especially because of this understanding of the role of human vacuolar protein Tsg101 on the proliferation of HIV in the human body, which has further deepened the research on HIV. Leonid et al. used poliovirus as a model, and used RNAi to induce intracellular immunity of cells and produce antiviral effects, especially for RNA viruses. For viruses that are susceptible to mutation, a variety of dsRNA targeting the conserved sequences of viral genes can be designed to reduce its resistance to dsRNA. Maen et al. also used RNAi technology to successfully block the function of an abnormally expressed nuclear transcription factor gene 21 associated with cell proliferation and differentiation in MCF-7 breast cancer cells. The application of RNAi technology can not only greatly promote the development of the human post-genome project (proteomics), but it is also possible to design RNAi chips to screen drug target genes with high throughput, and to detect the expression inhibition of the human genome one by one to clarify the genes. Function, and it will also be used in gene therapy, new drug development, biomedical research and other fields, using RNAi technology to inhibit abnormal gene expression, opening up new ways for the treatment of cancer, genetic diseases and other diseases.