Mechanism of action of RNAi and synthesis method of siRNA
RNA interference (RNAi) is a process in which a double stranded RNA (dsRNA) molecule shuts down or silences a corresponding sequence gene at the mRNA level. dsRNA can inhibit the expression of targeted genes of different types of cells, and the proteins expressed by the targeted genes are hardly detected by specific antibodies. Thus, RNAi technology is again known as knock out or gene silencing. RNAi is a typical post-transcriptional gene regulation method, also known as post-transcriptional gene silencing (PTGS) [1] .
1 Development process of RNAi technology
As early as 1990, botanist Jorgensen et al. used petunia to test the purple pigment synthesis gene into plants, and tried to inject more copies of the pigment gene into the plant to make the flowers more colorful. As a result, many flowers did not produce more beautiful flowers, but instead produced white flowers. Further analysis revealed that these transferred genes not only did not express themselves as proteins, but closed the expression of pigment-related genes in the morning glory. Since this inhibition by the introduction of a foreign gene was initially confirmed to occur at the post-transcriptional level, this phenomenon is called cosuppression [2] . In 1994, Cogoni et al. introduced the exogenous carotenoid gene into wild-type R. solani, and the endogenous carotenoid gene in transformed cells was also inhibited. They called the inactivated form of this gene to eliminate it. Or genetic suppression (quelling) [3] . In 1995, Dr. Guo [4] of Cornell University tried to block the par-1 gene expression of C. elegans by antisense RNA, and also used a sense RNA to make a control, trying to observe the control experiment. The phenomenon of enhanced gene expression was found, but it was found that both antisense and sense RNA blocked the expression of this gene, and Guo et al. could not explain the phenomenon. It was not until 1998 that Fire et al. [5] discovered that this was caused by Dr. Guo's contamination of double-stranded RNA in the experiment. They also demonstrated that the single-stranded RNA transcribed after purification was very weakly blocked by injection of nematodes, while the purified double-stranded RNA efficiently and specifically blocked the expression of the corresponding gene. . In 1999, experiments by Hunter [6] further validated the existence of RNAi. After they removed the dsRNA, they injected the sense or antisense ssRNA into the nematode eggs without any gene silencing. On the contrary, if the above sense and antisense ssRNA are mixed, the dsRNA obtained by annealing and renaturation can be injected into the nematode eggs to significantly produce gene silencing, which confirms that the RNAi effect proposed by Fire et al. is caused by dsRNA. First discovered in Drosophila in 2000, long dsRNA can be processed into short fragments of 21-23 nt by ribonuclease. In 2001, RNAi was also reported for the first time in mammalian cells. In 2002, it was demonstrated that short hairpin RNA (shRNA) can induce specific gene silencing in mammalian cells [7] . In 2003, the model animals were treated with RNAi technology for the first time [8] . The 2006 Nobel Prize in Medicine did not insist on the “conventions†that have been practiced for decades. The American scientists Andrew Phil and Craig Melo were awarded in recognition of their discovery of RNAi in 1998. Since then, extensive and intensive studies have been carried out in different species of organisms, and it has been confirmed that dsRNA-mediated RNAi is present in fungi, fruit flies, Arabidopsis thaliana, trypanosomes, planaria, leeches, zebrafish, mice. , rats, monkeys and even humans and other organisms [2] .
2 Mechanism of action of RNAi
The current hypothesis about gene silencing suggests that post-transcriptional gene silencing mainly includes the initial phase, the effect phase, and the multiplication phase.
2.1 Initial stage
Exogenous introduction or introduction of double-stranded ribonucleic acid (dsRNA) by transgenic, transposon, viral infection, etc., specifically binds to RNase III (RNAaseIII endonuclease) Dicer in cells, and dsRNA is cleaved A short-chain dsRNA with a 3'-end single-stranded tail and a phosphorylated 5'-end of 21 to 23 nt in length, ie, small interfering RNA (siRNA). (The following pictures are from Chen Li et al., "Progress in RNA Interference in the Field of Medicine")
2.2 Effect stage
The double-stranded siRNA can bind to a ribozyme complex containing Argonauto (Ago) protein to form an RNA-induced silencing complex (RISC) and be activated. In the case of ATP-supplied, the activated RISC separates the double strands of the siRNA, and the core component endonuclease Ago in RISC is responsible for catalyzing one of the strands of the siRNA to find a complementary mRNA strand, which is then cleaved. The antisense strand is first paired with a homologous mRNA, and then RISC cleaves the mRNA at a position 12 bases from the 3' end of the siRNA, thereby preventing target gene expression and silencing the gene [9] .
2.3 multiplication phase
Under the action of RNA-dependent RNA polymerase (RdRP), siRNA is used as a template and siRNA is used as a primer. Amplification generates a sufficient amount of dsRNA as a substrate to provide Dicer enzyme, which produces more siRNA and can form RISC again. And continue to degrade mRNA, resulting in a cascade amplification effect. And acting on the target mRNA. This is repeatedly multiplied, thereby further amplifying the action of RNAi. Therefore, a small amount of siRNA can produce efficient gene silencing [10] .
3 RNAi design and synthesis
3.1 siRNA design
To design high-efficiency siRNA, firstly, through the three gene sequence databases NCBI, DDBJ, and BMBL, the relevant gene sequences are searched to obtain targeted mRNA or cDNA sequences, and then siRNA is rationally designed. The design principle of conventional siRNA is based on mature mRNA. If the DNA sequence is used as a reference, the downstream sequence of the cDNA transcribed region +50 to +100 is preferred. The untranslated regions at both ends are not used as the basis for siRNA design. The sense strand and the antisense strand of the siRNA dimer each preferably contain 21 nt, and the 3' end is a prominent end. The 3' convex end is uridine (U), and the 5' recessed end is adenosine (A) mRNA sequence should be preferred. A Gen-Bank BLAST query was performed to ensure that the selected siRNA coding was not homologous to other genes [11] . Not all subunits of mRNA can form effective siRNA, about 60%~70% can exert silencing effect, because 5' and 3'-UTR have abundant regulatory protein binding regions, produce UTR binding protein or translate The initial complex may affect the RISC binding mRNA, thus affecting the effect of siRNA, so the silencing effect rate is quite different. It is necessary to design 3~4 siRNAs for a target gene to ensure that an effective sequence can be screened [12] .
3.2 siRNA synthesis method
The methods for preparing siRNA mainly include chemical synthesis method, in vitro transcription method, enzymatic digestion method, in vivo transcription method and the like. When the most effective siRNA has been found, it is suitable to synthesize two complementary RNA single strands of about 21~23 nt long by using a nucleotide monomer as a raw material, and then annealing to form a double-stranded complex. The obtained product has high purity, high interference efficiency and simple synthesis, but the preparation cycle is long, the action time is short, and the price is expensive, which limits its application and promotion [13] . In vitro transcription [14] uses two complementary DNAs as a template, and the T7 promoter is ligated upstream of the sense and antisense strands of the target sequence, and two single-stranded RNAs are obtained by in vitro transcription using T7 RNA polymerase. The two single strands are annealed to form double-stranded RNA, which is then digested with RNase, and the resulting product is purified to obtain the desired double-stranded RNA. This method is suitable for screening for effective siRNA, but not for long-term specific siRNA. the study. The enzymatic digestion method [15] is to first chemically synthesize a 200-1000 bp complete long-length dsRNA of the target gene in vitro, and digest it with the key siRNA enzyme Dicer or RNase III formed in the cell to obtain various different targets for the same purpose. The siRNA mixture at different sites of the gene, and then transfecting the mixture into the cells, can achieve higher inhibition efficiency. The method has high inhibition rate and saves time and labor, but the mixture produced by this method may lead to non-specific inhibition, and it is difficult to transcribe long-chain RNA in vitro, and the cost of Dicer enzyme is higher [16] , and this method is not suitable for long time. Research projects, or the need for a specific siRNA for research, especially gene therapy. In vivo transcription [17] is the transfer of a siRNA plasmid, a viral expression vector or a PCR product carrying a siRNA expression cassette into a cell, which is expressed by the cell to produce an RNA interference effect. The siRNA expression vector is commonly used as a vector containing an RNA polymerase III (polIII) promoter capable of expressing shRNA in mammalian cells [18] by transfecting a promoter U6 or H1 containing RNA polymerase II/III, and a downstream thereof. A small specific plasmid or viral vector is introduced into the host cell to transcribe shRNA, which is cleaved into siRNA by the Dicer enzyme in the cell. The advantage of this method is that it has strong cell specificity and is suitable for long-term studies of gene function [2] . siRNA expression cassettes (SECs) [17] is a siRNA expression template prepared by PCR, which can be directly transferred into cells for expression without prior cloning into a vector. PCR was carried out by primer extension to generate an expression frame containing a RNA polymerase III promoter U6 or H1, a short DNA template encoding the shRNA, and an RNA polymerase III termination site, and then directly transfected into the cells. This method provides a convenient tool for screening effective siRNA fragments and suitable promoters [2] . The main disadvantage is that the PCR product is difficult to transfect cells. If transfection reagent can improve the transfection efficiency of SECs, this method will be more widely used.
references
[1] FIREA, XU S, MONTGOMERYMK, et al. Potentand specific genetic interference by double stranded RNA in Caenorhabdits elegans [J]. Nature, 1998, 391: 806.
[2] Chen Li, Qin Wei, Zhu Yuanyuan. Progress in RNA interference research in the field of medicine[J]. Pharmaceutical Biotechnology, 2009, 16(1): 083~089.
[3] COGONI C, ROMANO N, GM Suppression of gene expression by homologus trans-genes [J]. Antonie Van Leeuwenhoek, 1994, 65(3): 205.
[4] GUO S, KEMPHEUSKJ. Par-1, a gene required for establishing polaring inelegansembryosenkinase thatasymmetrically distributed [J]. Cell, 1995, 81: 611.
[5] FIRE A, XU S, MONTGOMERY MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans [J]. Nature, 1998, 391 (6669): 806.
[6] HUNTER, PC Genetics: A touch of elegance with RNAi [J]. Current Biology, 1999, 9:440.
[7] PAUL CP, GOOD PD, WINER I, et al. Effective expression of small interfering RNA in human cells [J]. Nat Biotechnol, 2002, 20: 505-508.
[8] SHEN C, BUCK AK, LIU X, et al. Gene slience by adenovirus-delivered siRNA [J]. FEBS Lett, 2003, 539: 111-114.
[9] FIRE A, XU SQ, MONTGOMERY MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans [J]. Nature, 1998, 391 (6669): 806-811. [10] PD. Z. RNA interference: Listening to the sound of silence [J]. Nat Struct Biol, 2001, 8 (9): 746-750.
[11] ELBASHIR SM, HARBORTH J, WEBER K, et al. Analysis of gen function in somatic mammalian cells using a small interference RNAs [J]. Methods in Enzymology, 2002, 26: 199-213.
[12] DG Small silencing RNAs: state-of-the-art [J]. Adv Drug Deliv Rev, 2009, 61(9): 672-703.
[13] RM Small interfering RNAs and their chemical synthesis [J]. Angew Chem Int Ed Engl, 2002, 41 (13): 2265-2269.
[14] BRUMMELKAMP TR, BERNARDS R, RA A system for stable expression of short interfering RNAs in mammalian cells [J]. Science, 2002, 296: 550-553.
[15] MYERS JW, JONES JT, MEYER T, et al. Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing [J]. Nat Biotechnol, 2003, 21(3): 324-328. [16] SHREY K , SUCHIT A, MJ N. RNA interference: emerging diagnostics and therapeutics tool [J]. Biochemi Biophys Res Commun, 2009, 386(2): 273-277.
[17] Yuan Chengliang, Jin Xiaotong. Progress in RNA interference principle and application research [J]. Western Medicine, 2009, 21 (4): 664-666.
[18] MIYAGISHI M, KT U6 promoter2driven siRNAs with four uridine 3'overhangs deliverative inhibitor targeted gene expression in mammalian cells [J]. Nat Biotechnol, 2002, 20(5): 497-500.
Pain Relief Patch for neck shoulder, lower back and leg.
[Name] Medical Cold Patch
[Package Dimension] 10cm×12cm 2pieces/box
The pain relief patch is composed of three layers, namely, backing lining, middle gel and protective film. It is free from pharmacological, immunological or metabolic ingredients.
[Scope of Application] For cold physiotherapy, closed soft tissue only.
[Indications]
The patches give fast acting pain relief for strains, sprains, cramp, bruises, swollen areas or joint stiffness.
[How To Use a Patch]
Tear off the packaging bag and remove the patch. Remove the protective film and apply the patch on the skin. One piece, one time. The curing effect of each piece can last for 8-12 hours.
[Attention]
Do not apply the patch on the problematic skin, such as wounds, eczema, dermatitis,or in the eyes. People allergic to herbs and the pregnant are advised not to use the medication. If swelling or irritation occurs, please stop using and if any of these effects persist or worsen.notify your doctor or pharmacist promptly. Children using the patch must be supervised by adults.
[Storage Conditions]
Store below 30c in a dry place away from heat and direct sunlight.
Pain Relief Patch For Neck Shoulder,Lower Back And Leg
Pain Relief Patch,Pain Relief Pad,Pain Relief Plaster,Neck Pain Relief Patch,Back Pain Relief Pacth
Shandong XiJieYiTong International Trade Co.,Ltd. , https://www.xjplaster.com
