RNA interference is the mechanism by which aberrantly expressed double stranded RNA (dsRNA) suppresses the expression of a gene bearing its complementary sequence. This is referred to as gene silencing or gene knockout. RNAi naturally occurs against aberrant self and viral genes. It has been observed that if 21-25 nucleotide long siRNA is introduced mechanically, it can cleave target mRNA. The successful implementation of this technology requires the design of double stranded RNA (siRNA) which is identical to the specific regions of the target genes. RNA interference is a phenomenon whereby small interfering RNA strand (siRNA) inhibit the gene expression at the level of transcription or translation of specific genes (PTGS). RNAi is a defence mechanism against viruses and is important in annotating genes, regulating development and genome maintenance. RNAI is mediated by small interfering RNA i.e. siRNA which is sometimes also known as short interfering or silencing RNA.
Silencing RNA is a class of double stranded RNA molecules which is about 20-23 nucleotide long and play a significant role in the life sciences. This significant discovery was done by David Baulcombe's group in England and Norwich as a part of post-transcriptional gene silencing (PTGS) study in plants. The synthetic siRNA were shown to be able to induce RNAI in mammalian cells by Thomas Tuschl, et al.
The work flow of siRNA continues as said. The long double stranded RNA or siRNA (24bp) are introduced which afterwards enter a cellular pathway commonly referred to as the RNA interference (RNAi) pathway. The double stranded RNA get processed into 20-25 nucleotide small interfering RNA by an RNAse III enzyme (Dicer). The siRNA assemble into endoribonuclease containing complexes known as RNA- induced silencing complexes (RISCs). Activated RISC then binds to the complementary transcripts by base pairing interactions between siRNA antisense strand and mRNA. The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing.
RNAi acts as a tool for the functional genomics. RNAi i.e. gene silencing or gene expression down-regulation is the process whereby a dsRNA induces the homology dependent degradation of cognate mRNA. The resulting technology provides a powerful tool for biological researchers by allowing a gene to be virtually knocked out inside the cell through the introduction of synthetic or plasmid-derived siRNA. This process is known as reverse genetics and is similar to assessing the function of small numbers of genes through the production of knock-out mice. Since 2006, success has been achieved in engineering virus resistance using siRNA which requires synthesis of well conserved siRNA that can initiate RNAi mediated silencing of critical viral genes.
The guidelines of designing siRNA are as follows:-
i) Select target regions from 50 to 100 nucleotide downstream of the start codon.
ii) Choose the target site starting with NAR(N17)YNN, where N is any nucleotide, R is purine (A,G) and Y is pyrimidine (C,U), for e.g. AA(N19)TT.
iii) Nucleotides 1 to 19 of the sense small interfering RNA strand correspond to the position 3-21 of the 23 nucleotide target motif.
iv) The target sequence is selected to have around 50% of the GC content.
v) The selected siRNA sequences should be aligned against EST libraries to ensure that only one gene will be targeted.
vi) Avoid the stretches of more than four adenine in the target sequence because they act as termination signal for RNA polymerase III.
vii) Eliminate from the consideration any target sequence with more than 16-17 continuous base pairs of homology to other coding sequence i.e. off target cleavage.
There are several tools and programs to design siRNA, i.e., siDESIGN Centre, siRNA target finder, siRNA design, Jemboss tool, etc. Thus, siRNA are designed against several viral genes.
The synthesis of suggested 21-24 base pair long siRNA from siRNA manufacturers. Introduce these siRNA into the host cell. The inoculants must be checked to test the efficiency of the putative siRNA. Selection of the transformants can be done by using various molecular biology techniques, i.e., western blotting to justify the presence of any viral protein against which we have designed the siRNA. RT-PCR (Reverse Transcriptase PCR) to look for the presence of cDNA band of the viral gene using specific primer.
Though, there are always some ethical issues associated with these technologies finding its platform in today's life. But this is the bright part in case of siRNA as it is not resulting into any transgenic product. The integrity of the host genome is never disturbed as the synthetic double stranded RNA used initiate the RNA mediated silencing without integrating in the host genome. The process is systemic, broad spectrum and self amplified. The double stranded RNA is quite stable, easily introduces and without any expression vector and range of regulatory elements.
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