Progress in RNAi technology research

RNA interference (RNAi) is a phenomenon in which multiple enzymes are mediated by double-stranded RNA (dsRNA) in a variety of organisms. The RNAi phenomenon has been discovered in different organisms, which botanists call co-suppression or post-transcriptional gene silencing (PTGS); researchers of nematodes and fruit flies call it RNAi; fungi Researchers call it a quelling. It has now been recognized that RNAi is ubiquitous in organisms and may have a common biological significance and a similar mechanism of action.

1. Discovery and development of RNAi

In 1990, in the study of the transgenic plant Petunias, it was found that the pigment synthesis gene was placed in a strong promoter and introduced into the morning glory to try to deepen the purple color of the flower. Expression, and self-pigmentation is also attenuated. This phenomenon was called PTGS or "cosuppression" at the time. After Ramamor N introduced a synthetic carotenoid gene into Neurospora crassa, about 30% of the genes in the transformed cells that are self-synthesizing carotenoids are inactivated. They are called gene quilling. When Corn S. et al. used antisense RNA technology to block the expression of parl gene in C. elegans, it was found that both sense RNA and antisense RNA can suppress gene function expression, and the roles of the two are independent. The mechanism is different. For the first time, Fire A et al. of the Carnegie Institution of Washington demonstrated that the phenomenon belongs to post-transcriptional gene silencing, and this phenomenon is called RNA interference. In 2006, it won the Nobel Prize in Physiology and Medicine. In the short span of 1999, RNAi was found to be widespread in almost all eukaryotic cells from plants, fungi, nematodes, insects, frogs, birds, rats, mice, monkeys, and humans. In 2000, it was discovered that RNAi was also present in mouse early embryos and in E. coli.

2. The mechanism of action of RNAi

Hamilton et al. and Ketting et al. identified approximately 25 bases of RNA complementary to the sense and antisense strands of a silenced gene in co-suppressed tobacco and tomato, respectively. Zamore et al. found that double-stranded RNA injected into Drosophila cells was cleaved into a double-stranded RNA fragment of 21 to 23 bases in length. They believe that these siRNAs play an important role in the RNAi process. These siRNAs generally have two prominent bases at the 3' end of the double strand of 21 to 23 base pairs, most of which are uracil, thymine and have a hydroxyl group; and the 5' end is phosphorylated. These characteristics may be that they are distinguished from other RNAs and can be specifically recognized, and this feature is also a feature of RNase III digestion. Studies have shown that mRNA is specifically cleaved in the cytoplasm. When double-stranded RNA enters the cell, it binds to RNase III-like Dicer and is subsequently cleaved into 21 to 23 base siRNA. The siRNA binds to RISC (RNA induced silencing complex), and if the 5'-end of the double strand is not phosphorylated, its phosphokinase activity is initiated, phosphorylated, and uncoiled. The antisense strand RNA binds to RISC and activates its endonuclease activity. The activated RISC searches for cytoplasmic mRNA that is homologous to the antisense RNA, and is located at the center of the double strand, 10th from the 5' end of the double-stranded RNA. The base is cleaved to degrade the mRNA. Sijen et al. believe that the role of double-stranded RNA in the early stage of RNAi is to provide primers for RdRP (RNA dependent RNA Polymerase) and to synthesize more double-stranded RNA using the target mRNA as a template. This hypothesis is confirmed in other species.

Several RNAi-related genes have been identified in species such as nematodes, fruit flies, Arabidopsis, and cytobacteria, such as the nematode genes ego-1, rde-1 and rde-4, and the qde of the bacterium -2, the ago1 gene of Arabidopsis and the like. RNAi is quite conservative in the evolution and development of organisms. AGO1, QDE2 and RDE1 play a similar role in plant transgene silencing, fungal repression and animal RNAi. Boutla et al. extracted RNA from plants that produced gene silencing and caused specific gene silencing after injection into nematodes. However, there are still large differences in RNAi between different species. The use of 21 base pair double-stranded RNA in mammalian cells can elicit a specific inhibitory response, while longer double-stranded RNA (more than 30 base pairs) transfects mammalian cells can cause non-specific gene suppression. The long double-stranded RNA activates the protein kinase PKR, which activates the translation initiation factor eIFa through a series of phosphorylation, resulting in translational repression or activation of RNase L by long dsRNAs, resulting in non-specific RNA degradation.

3. RNAi technology

The first thing to do with RNAi is the selection of the gene of interest. In the experiments currently performed, the sequence of interest corresponding to the phenotype that can be easily detected is generally selected. Since the discovery of the penetrance using the cDNA template RNAi mutation phenotype is higher than the use of the genomic template, the cDNA sequence becomes the designated template. However, exon-rich genomic sequences can also be used in the absence of cDNA. RNAi efficiency may be affected by other factors such as the length of the dsRNA used and structural analogs. Methods of delivering dsRNA may also affect the strength of the RNAi response. The most common method of delivering RNA in C. elegan is microinjection of microbial polynuclear bodies, body cavities or viscera. Microinjection of adult worms in C. elegan is considered to be the most efficient RNAi pathway. Although other methods of delivering RNA are less intense, dsRNA soaking and feeding to worms indicates that the E. coli method of C. elegan dsRNA has been used for whole genome functional analysis and high throughput screening.

4, the application of RNAi

4.1 Studying Functional Genomics

Although there are still many unclear mechanisms of action of RNAi, many molecular biologists believe that RNAi is a very useful tool for studying functional genomes. Because RNAi has been used to study and clarify the functions of genes including Drosophila, C. elegans and some plants. Recently, researchers have even found a more convenient way to induce stable sequence-specific silencing by enhancing the expression of endogenous RNA hairpin structures, thereby establishing a cell line that continuously lacks a functional phenotype. It provides a large number of silent cells for biochemical analysis, and it is also possible to perform long-term observation and evaluation of a certain phenotype. It is expected that in the near future, many types of cultured cells will be grown on the "RNAi chip", the dot matrix of siRNA, which can record the phenotype after each gene in the genome is inhibited and the genes are gradually suppressed. The comprehensive phenotype makes it possible to know the function of genes and the interactions between them. In conclusion, RNAi will serve humans in the post-genomic era in order to solve the disease mechanism and identify key issues in drug-targeting, gene function identification, which is simple and efficient.

4.2 Applications in developmental biology and nuclear transfer

In order to study the events of embryos during early development, it is usually thought to remove the expression of specific genes. If the function of genes of interest in embryonic cells can be removed at a specific time, people will get valuable. Information.

When Dicer's homolog SIN1/CARPEL FACTORY is mutated, the animal individual will develop severe developmental defects, indicating that the RNAi pathway can regulate the expression of development-related genes in a sense. Recent data indicate that Dicer is required for the production of single temporary RNA (stRNA), stRNA can inhibit translation of target mRNA, and PPD proteins containing PAZ and PIPW domains are involved in stem cell, egg production, and tissue differentiation. It plays an important role. The above data indicate that Dicer and its homologues are indeed critical during animal development. A fertilized egg or animal has the same genetic information in its somatic cells, so it can differentiate into various types of cells due to the expression of different genes.

Experiments have shown that the dominator and guardian of the Dicer genome exists in the cytoplasm of mammals. The nuclear transfer produces cloned animals to remove a part of the cytoplasm, that is, to remove a part of Dicer and mRNA, then this step affects The reprogramming of donor nucleus genes, Dicer in the initial developmental regulation of somatic clonal animals: from the post-transcriptional silencing of somatic nucleus to the emergence of zygote nuclear transcriptional activity, what role is still to be studied. In addition, the composition of the RNAi action and the imprinted gene, as well as the relationship between the X chromosome inactivation, are very attractive research topics.

4.3 gene therapy

Target recognition of RNAi is a highly sequence-specific process. From the siRNA to the target strand, the complementary target recognition is specific, and the penultimate nucleotide in the 3' end of the projection affects the cleavage of the target mRNA. Once mismatched, the effect of RNAi will be reduced by 2 to 4 times. The 5' and 3' of the guide siRNA are also relatively permissive for mismatch, but the nucleotide at the center of the siRNA as opposed to the cleavage site of the target RNA is a very important factor in determining specificity, even There is a nucleotide change, and RNAi cannot be detected during the experiment, indicating that the siRNA duplex can recognize the mutation or polymorphic alleles of the gene target, which is very beneficial for future treatment development. It is not known whether RNAi-resistant proteins encoded by viral or animal viral genes inhibit RNAi, but understanding the mechanism of action of RNAi systems and their interaction with animal viruses is to establish RNAi-based human disease gene therapy. An important part of it.