siRNA or small interfering RNA is a non-coding RNA molecule. It is a double-stranded RNA molecule and is also called silencing RNA due to its involvement in silencing or inhibiting the expression of a gene. It controls transposons and helps in fighting viral infections.
siRNA was first discovered in plants by David Baulcombe’s group, for their role in post-transcriptional gene silencing (PTGS). siRNA has a therapeutic potential to cure many genetic diseases due to its ability to inhibit the expression of a specific gene.
Various small non-coding RNAs have been discovered recently that have a role to play in the regulation of gene expression, RNA processing, transcription, translation, RNA stability, etc. E.g. microRNA (miRNA), small interfering RNAs (siRNA), piwi-interacting RNA (piRNA), etc.
siRNA is a double-stranded RNA molecule. It is around 20-25 nucleotides long. It contains 5’ phosphorylated ends and 3’ hydroxyl groups. It has two nucleotide overhangs on both strands at the 3’ end.
Mechanism of Action
It regulates the expression of a coding gene. The process by which it inhibits the expression of genes is known as RNA interference or RNAi. It is involved in post-transcriptional gene silencing (PTGS).
RNA interference or RNAi is a process by which a dsRNA molecule binds and prevents the translation of mRNA having a complementary sequence. It induces gene silencing. The mechanism of RNA interference was discovered by Fire and Mellow in 1998.
The steps involved in the gene silencing by RNA interference are as follows:
- The first step is the formation of siRNA from a longer dsRNA molecule. The longer dsRNA is cleaved by an endoribonuclease known as Dicer. The siRNA generally have two nucleotide long overhangs at the 3′-end of both strands.
- Then siRNA is associated with other proteins to form the RNA-Induced Silencing Complex (RISC).
- siRNA then unwound to form a single-stranded siRNA. The sense strand is cleaved and the antisense strand becomes part of the active RISC complex.
- The single-stranded siRNA that remains the part of the active RISC complex finds the target mRNA.
- After finding the complementary mRNA, siRNA binds to the target mRNA and cleaves the mRNA by the action of catalytic RISC proteins.
- It leads to degradation of the mRNA and translation of mRNA is inhibited. Thus, the gene is silenced.
Function and Application
It is involved in cellular defence. It controls the damage by transposons and viral infections.
siRNAs silence genes at the post-transcriptional level. They cleave mRNA molecules with a sequence complementary to the siRNA molecule and thereby stop the translation process or gene expression.
Expression of any gene can be interfered with using siRNA having complementary sequences. This can be utilised in drug development to regulate gene expression by introducing siRNA into the cell.
siRNA is useful in research. It can also be used to study the function of a specific gene.
siRNA can be used to treat various diseases such as cancer.
Delivering the siRNA for therapeutic use is a challenge. It needs to reach the target site without being degraded.
The delivery of siRNA in the cell can be viral-mediated or non-viral. The non-viral delivery involves electroporation or transfection. In the transfection, siRNA is delivered using polymers, liposomes, nanoparticles, lipid conjugation, etc.
The naked chemically modified siRNA delivery is found to be efficient in the lungs and brain. The synthetic siRNA can be degraded by enzymes therefore the gene silencing effect is short-lived. Therefore, delivering the siRNA using a DNA template is found to be more effective.
The viral-mediated delivery can be done using recombinant viral vectors using retrovirus, adenovirus, etc. A virus having a complementary RNA genome or transposon can be used to infect cells. The problem with this type of delivery is that it can trigger an immune response against viruses.
Use of RNAi for Pest Resistant Plants
RNA interference can be used in transgenic plants that can protect themselves from a parasite infection. E.g.
The root of tobacco is infected by the nematode Meloidogyne incognita and greatly affects the yield. To prevent the infection RNAi method is used.
- The nematode-specific genes were delivered to the host cell by using the Agrobacterium vector using recombinant DNA technology.
- This triggered the formation of both sense and antisense strands of RNA from the inserted DNA.
- The sense and antisense strands of RNA produce dsRNA and initiate the RNA interference process.
- The nematode-specific interfering RNA attacked the specific mRNA of the nematode leading to silencing of the gene and the nematode could not survive in the host.
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