Sirna Notes

Small interfering RNA (siRNA) is a double-stranded RNA molecule that is non-coding. It is also referred to as silencing RNA due to its role in suppressing or blocking gene expression. Additionally, siRNA is involved in controlling transposons and aiding in the defense against viral infections.

siRNA was first discovered in plants by David Baulcombe’s group, for their role in post-transcriptional gene silencing (PTGS). It has since been found to have therapeutic potential to cure many genetic diseases, due to its ability to inhibit the expression of a specific gene.

siRNA Structure

Recently, a variety of small non-coding RNAs have been discovered that are involved in the regulation of gene expression, RNA processing, transcription, translation, RNA stability, etc. Examples of these include:

  • microRNA (miRNA)
  • small interfering RNAs (siRNA)
  • piwi-interacting RNA (piRNA)

siRNA is a double-stranded RNA molecule of approximately 20-25 nucleotides in length. It is characterized by 5’ phosphorylated ends and 3’ hydroxyl groups, with two nucleotide overhangs on both strands at the 3’ end.

Mechanism of Action

RNAi is a process by which a coding gene’s expression is regulated. It is involved in post-transcriptional gene silencing (PTGS), and is also known as RNA interference.

RNA interference (RNAi) is a process that was discovered by Fire and Mellow in 1998, in which a double-stranded RNA (dsRNA) molecule binds to mRNA having a complementary sequence, thus preventing its translation and inducing gene silencing.

The Steps Involved in Gene Silencing by RNA Interference Are:

The longer dsRNA molecule is cleaved by an endoribonuclease known as Dicer to form siRNA, which usually have two nucleotide long overhangs at the 3’ end of both strands.

The siRNA is then combined with other proteins to create the RNA-Induced Silencing Complex (RISC).

The single-stranded siRNA is then cleaved, with the sense strand being discarded and the antisense strand becoming part of the active RISC complex.

The single-stranded siRNA which is a part of the active RISC complex locates the target mRNA.

After finding the complementary mRNA, siRNA binds to the target mRNA and cleaves it through the catalytic activity of RISC proteins.

The degradation of the mRNA and inhibition of its translation results in the silencing of the gene.

Function and Application

It is involved in cellular defence, controlling the damage caused 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.

siRNA can be used to regulate gene expression in drug development by introducing siRNA with complementary sequences into the cell, thereby interfering with the expression of any gene.

siRNA is a useful tool for research, as it can be used to study the function of a specific gene.

siRNA can be used as a potential treatment for various diseases, including cancer.

Delivering siRNA for therapeutic use is a challenge, as 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 delivery of naked chemically modified siRNA is found to be efficient in the lungs and brain, however, the gene silencing effect is short-lived as synthetic siRNA can be degraded by enzymes. Therefore, delivering the siRNA using a DNA template is more effective.

The delivery of genetic material to cells via viral vectors can be accomplished using retroviruses, adenoviruses, etc. Viruses with a complementary RNA genome or transposon can be used to infect cells. However, this type of delivery can cause an immune response against the viruses.

Using RNAi to Create Pest Resistant Plants

Transgenic plants that can protect themselves from a parasite infection can be developed through the use of RNA interference (RNAi). For example:

The root of tobacco is infected by the nematode Meloidogyne incognita, which greatly affects the yield. To prevent the infection, the RNAi method is used.

The Agrobacterium vector was used to deliver the nematode-specific genes to the host cell through recombinant DNA technology.

The insertion of DNA resulted in the formation of both sense and antisense strands of RNA.

The sense and antisense strands of RNA produce double-stranded RNA (dsRNA) and initiate the RNA interference (RNAi) process.

The nematode-specific interfering RNA targeted the specific mRNA of the nematode, resulting in the gene being silenced and the nematode being unable to survive in the host.

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