Research progress of RNAi

邹建

【Abstract】the double stranded RNA into cell could cause homologous gene silencing， a phenomenon called RNA interference （RNA interference， RNAi）. Research progress of RNA interference characteristics， in this paper， the mechanism of RNA interference technology， RNA interference and existing problems are summarized.

【Key words】RNA interference； double stranded RNA； gene silencing； gene therapy

1.the definition of RNAi

RNA interference （RNA interference， RNAi） [1] is composed of double stranded RNA （double stranded RNA， dsRNA） molecules close process corresponding sequence of gene expression or the silent at the mRNA level. DsRNA can inhibit the cell types targeted gene expression， using antibodies specific hardly detected target to gene protein expression. Therefore， RNAi technology is called gene knockout （knock out） or gene silencing （gene silencing）. RNAi is a typical post transcriptional gene regulation method， also known as post transcriptional gene silencing （post transcriptional gene silencing， PTGS）. RNAi phenomenon exists widely in biology. It is called quelling in fungi， in Arabidopsis， tobacco and other plants called PTGS or co suppression （cosuppression）， in invertebrates if fly， Hydra， nematode and vertebrate zebrafish， mouse， in the animal world is called RNAi. PTGS may be a biological community， including an ancient， conserved and ubiquitous plant and animal extremely important genetic behavior.

RNA interference is a protective mechanism against the invasion of foreign genetic elements in genomes of transposon or virus and the like， has the following 4 important characteristics： ① RNAi belongs to the post transcriptional gene silencing （PTGS） mechanism， research to date that does not produce any effect of RNAi on DNA sequence of chromosome replication and transcription processes； the sequence specificity， recently synthesized siRNAs （in short RNA， is a direct role in the process of molecular RNi） show into a host cell experiment， even though the siRNAs is only one base mismatch with the target sequence， also can make the interference effect greatly weakened； the effective inhibition of gene expression the phenotype， can cause the gene deletion mutant； but far less than the number of endogenous mRNA dsRNA can realize complete inhibition effect， i.e. the process exists signal positive feedback amplification effect； effect of the RNAi can be in different cells of long distance transmission and maintain， and in some species （such as the nematode） a hereditary.

2.On RNAi found

In 1995， Dr. Guo[2] of Cornell University in the experiment by antisense RNA blocks Caenorhabditis elegans （C.elegans） expression of the PAR-1 gene， at the same time， she also just do a RNA to control， was observed in the control group gene expression enhancement phenomenon， but only to find that the antisense RNA He Zhengyi blocked the expression of the gene the. Guo et al have been unable to explain the phenomenon. Until 1998， Fire[1] found that this is due to Dr. Guo in the experiment of pollution caused by double stranded RNA （single strand RNA they have shown that transcription obtained after purification by injection of nematode blocking effect is very weak， while the double stranded RNA purified can block highly specific expression gene， their corresponding this is called RNA interference. Effect of RNAi potential led Fire and Timmons to experiment， will be able to express and double chain RNA C.elegans unc-22 gene homologous to the bacterial feeding nematode， nematode showed similar results with unc-22 deletion phenotype of [3]. Subsequent experiments showed that the nematode dip in RNA can also induce gene silencing. Loss of function mutants of this technology makes the large-scale screening of nematode RNAi induced become possible， and has led to a subsequent large for this model of biological gene knockout studies. Currently the application functions of RNAi technology on approximately 19000 genes in C.elegans almost all forecasts in genome research.

In 1999， Hunter[4] 's experiments verify the existence of RNAi. They will be the removal of dsRNA， then the sense or antisense ssRNA injection nematode eggs， did not produce the phenomenon of gene silencing. On the contrary， if the sense and antisense ssRNA mixture， by annealing the refolded dsRNA injection can produce significantly gene silencing nematode eggs， which confirms the role of RNAi proposed by Fire is caused by dsRNA conclusion.

3.Mechanism of action of RNAi

3.1 the function of RNAi is embodied in two levels

The transcription level. RNA methylation mediated DNA （RNA-directed DNA methylation， RdDM） virus in a plant was first discovered.

DsRNA （double-strand RNA） were degraded into small fragments of 21-23 nucleotides long RNA， these small RNA molecules can be DNA methylation induced by [5] homologous sequences in the nucleus. Signal and RNA-DNA methylation in the sequence specific binding of [6]. When the dsRNA containing promoter sequences homologous， can make the homologous target methylation of promoter sequences， so that the target promoter lost their function， causes the downstream gene silencing. Non pathogenic virus caused transcriptional gene silencing related RdDM first found in cauliflower mosaic virus 35S promoter， which contained nopaline synthase promoter （NOSpro） sequence of NOSproRNA. In transgenic tobacco， according to reports not adenylation methylation NOSpro dsRNA can induce the homologous to NOSpro DNA， and make a copy at the transcriptional level trans inactivation. In the chain cell Pythium， DNA methylation may be the methylation of siRNA mediated heterochromatin mediated histone H3 K9， is catalyzed by H3 methyltransferases [7].

The post transcriptional level. The specific cutting of dsRNA RNA enzyme III family nuclease Dicer dependent ATP cleavage of dsRNA， which is decomposed into a 2 nucleotide 3 'overhang end （overhang） of 19 ～ 21b small segments of double stranded siRNA. （2） RISC （RNA induced silencing complex RNA-induced silence complex） recognition and degradation of mRNA. RISC is a kind of protein -RNA effector nuclease complex. Double stranded siRNA as an important component of the RISC dependent ATP helicase， which leads to the activation of RISC， and then through the Waston-Crick mRNA base pairing substrate recognition and binding， and since the siRNA 3 ′ end cuts mRNA to less than 12nt fragments. The degradation of [8. To test that some human tissue factor， different sites of several different siRNA can attack with a mRNA. Only a portion of the siRNA will result in gene silencing significantly， which indicates that the siRNA in the mRNA binding sites may be small， the siRNA and the inactive and active siRNA reversible competitive [9].

3.2 miRNA

Dicer enzyme produces two kinds of function specific small molecule RNA：siR2NA and miRNA （micro-RNA）. Tuschl in the Drosophila embryo extracts from long dsRNA processing by siRNA at the same time， the 16 20 - 23 nucleotides in size of short single stranded RNA. The RNA encoded by the Drosophila genome and expressed in 0 - 2 week embryo. They have the potential to regulate gene expression， known as miRNA[10]. About 21 nucleotides in size miRNA， as a single structure， the majority of miRNA and substrate mRNA incomplete pairing with their stability， miRNA does not change， but the inhibition of translation to make the gene silencing of [11]， but there is at least a plant miRNA （miR171） perfectly matched with the substrate mRNA， suggesting the possibility of the expression of miRNA gene by [12] the RNAi pathway regulation. Unlike the siRNA， because the miRNA is a single chain structure， which does not need the presence of ATP.

3.3 Amplification of RNAi

Due to the RNAi mechanism is extremely significant in the biological effects， it has been suggested， RNAi pathway should have corresponding amplification steps. Methods： ① amplification possible through the replication dsRNA or siRNA. Nishikura put forward "the transition of RNAi （transitive RNAi）" mechanism， the upstream sequence of target sequences from the original extension of the new siRNA， continue to degrade the homologous gene family members or cutting mRNA[13]. The RISC effect of the multi wheel. The RdRP （RNA induced RNA polymerase）： role of plants and Samsonite Rhabditis dsRNA induced silencing are similar to RdRP and proteins involved in [8]. General RNAi play a role in somatic cells， ego21 expression in germ cells known as a "zoom in model of random degradation of PCR" is suggestive of RdRP guided by siRNA chain identification combined with mRNA， Dicer enzyme substrate to produce a double stranded RNA， generated by siRNA more.

4.Methods RANi

Methods RANi synthesis in vitro and in vivo are synthetic siRNA method. The method of siRNA into the cell is divided into microinjection， electroporation， immersion method， engineering bacteria feeding method， transgenic method and virus infection method etc..

Methods Harborth [14] 21nt siRNA design synthesis in vitro is： in the gene library for targeting mRNA gene sequences， looking for AA + N19 + UU sequence or AA + N19 sequence from the start code after the seventy-fifth base and nucleotide sequence of mRNA， where N19 is an arbitrary 19nt； then the design of the selected sequences the G+ to C ratio of 30%--70%； to determine the sequence of Blast EST gene library search， identify gene targeting is the only； 21 antisense RNA sequence， and use SiACE2RNAi method for the synthesis of two strands of RNA， in the two chain of 3 'end of the last 2 nucleotides design and synthesis of dTdT sequence， in order to reduce intracellular degradation. SiRNA 21 nucleotides they using in vitro synthesized composition were transfected into human and rat cells， acting on NumA 16 gene， the expression of these genes was inhibited.

In 1998， Fire[1] by microinjection in vitro synthesized dsRNA injection to worms， observed the dsRNA from the injection of cells to spread to other cells in the body， and the RNAi effect can be inherited to the next generation， in the offspring still inhibit target gene expression. And feed the larvae can expression of double stranded RNA bacteria， or the larva soaking in the solution containing dsRNA， can also be observed mutant phenotype specific.

Because the RNA is easy to be RNA enzyme decomposition， and no matter the biosynthesis and chemical synthesis of dsRNA， costs are high and time-consuming， which restrict the application of RNAi technology in a certain extent. Synthesis of siRNA. Synthesis of siRNA induced by RNAi technology within the cell， can greatly reduce the cost， increase the operational experiment.

Miyagishi[15]， established U6 promoter in vivo siR2NA synthesis method of driving， successfully inhibited the expression of target gene in mammalian cells. They designed and built the synthesis of justice chain two U6 startup siRNA respectively 19nt and antisense strand， two RNA annealing in cells connected into double stranded RNA. Sui also establishes the control of U6 promoter， siRNA expression plasmid， unlike the siRNA template for the palindrome structure. In the downstream of U6 promoter sequence is siRNA 21nt coding sequence， 6nt interval sequence， reverse siRNA21nt coding sequence and is composed of 5 T oligonucleotide tail transcription termination sequence. This plasmid induced synthesis of RNA hairpin double chain structure. Expression of hairpin double stranded RNA than by the sense and antisense strands in cells after annealing the synthesis of dsRNA seemed to be able to effectively suppress the target gene. Brummelkamp[16] to construct the pSU2PER plasmid vector， using synthetic polymerase III promoter of H12RNA gene from RNA. The template design， the in vivo synthesis of siRNA showed a hairpin like structure， and can be modified 3 'end to form two uridylic acid projecting structure， similar to the siRNA structure of chemical synthesis. In order to make RNAi technology become can induce gene silencing control， Miyagishi has also established a tetracycline regulated U6 promoter system， through the control of U6 promoter can command RNAi occur at a particular time. And the clone screening， with intracellular expression system of siRNA cell line can long keep and use at any time to recovery. In addition， some play a key role in the development process of animal genes， if at the DNA level using gene knockout or mutation of modified genetic， it may be premature to gene silencing， produce lethal phenotype， the research can not continue. Expression of dsRNA is controlled by specific promoter， can be the destination at a specific time to start RNAi. Control of RNAi started in the initial stage of development， can study the early development of key genes in the later stage of development function.

Although the plasmid mediated RNAi has above-mentioned advantages， but DNA transfected with transient expression， the longest but a few days. And in some types of cells， transfection rate is very low， even in the same cell， transfection rate is not the same. Therefore， reverse transcription virus and adeno-associated virus [17， 18] is also used as a carrier. The principle is similar， are carrying the promoter and DNA template directed synthesis in cells， siRNA. The difference is the synthesis of siRNA virus vector mediated more fast， consistent and stable， even in some non differentiation， primary cultured cells， transfection rate can reach 100%.

Eric first will guide the hairpin structure of siRNA synthetic oligonucleotide sequence was cloned into pBS/ U6 vector， then the cutting down of U6 promoter and oligonucleotide sequences complementary to the target gene of the enzyme solution， reinforcing the sticky ends with Klenow， and then inserted into pMSCVpruo. The application of this retroviral vector， they successfully suppressed a human serine / threonine kinase （NDR） gene expression. Changxian will start H1-RNA primer oligonucleotide sequences cloned sequences and coding P53 gene of plasmid pShuttle to transport non promoter sequences， and then pShuttle253 and pAdEasy-1 recombination， obtained two recombinant adenovirus DNA， AdH1-53 and AdH1-empty. They also successfully observed P53 deletion phenotype induced by AdH1-53.

Phage promoter transcription technique has also been applied to the production of RNAi. Liang[8] proved the transcriptional activity of constitutively bacteriophage lambda pL promoter increased by 2 - 3 times. LaCount also use bacteriophage T7 promoter induced by green fluorescent protein gene silencing.

Another new development is the study of regulatory elements of 5 'UTR and 3' UTR. In the past， the sequence design of dsRNA is usually the target gene exon sequences， dsRNA containing the intron and promoter sequences generally cannot generate RNAi. But the recent discovery of RNAi can function in the 5 'untranslated region （5' UTR） or 3 'UTR[20， 21]， target gene silencing. Doench found in mammalian tissue culture， small interfering RNA and target mRNA 3 'UTR complementary sequence binding， inhibit the expression of endogenous miRNA， which encoding proven function is similar. Eckmann found RNA binding protein FBF by binding to regulatory elements fem-3 3 'UTR， influence the formation of nematode sperm， which decided the fate of the eggs， and inhibit the expression of sex determining gene. Yokota found in HCV genome， 5 'UTR is a highly conserved region， which make it an ideal target for siRNA. They designed siRNA targeting HCV genome with 5 'UTR， inhibit viral protein synthesis. These findings enable scientists to analysis are included in the 5 'UTR and 3' UTR in regulating device function. As the process of embryonic development was entirely dependent on mRNA and protein in the correct position and time expression or expression inhibition. Using green fluorescent protein tagged promoter or interested gene 3 'UTR， the gene and protein expression of real-time tracking of the position is more simple， do not need to have the in situ hybridization injury tissue fixation process and specific antibodies to the organization.

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