“The siRNA or small interfering RNA is a 22 to 25 base pair long smaller molecules of dsRNA having a dinucleotide overhang at the 3’ end, interfere in the protein synthesis by blocking the translation.”


 The presence of double-stranded RNA in a cell is a sign of danger. As the main three types of RNA in us are single-stranded. The dsRNA is not found in our cells because our genetic material is DNA not RNA. The RNA is the genetic material of some retroviruses thus, 

If dsRNA is present, it is a sign of infection, this will infect the cells and can cause cell death. 

Note: the ribosomal RNA contains some amount of dsRNA and some hairpin RNA molecules too. 

RNA is a type of nucleic acid that is present in the nucleus of a cell. Although it is not a genetic material in eukaryotes, some of the viruses known as retroviruses have RNA as their genetic material. 

DNA is the genetic material in all eukaryotes and prokaryotes, except retroviruses. The RNA is a ribonucleic acid made up of ribose sugar instead of deoxyribose sugar of the DNA. For more detail, on RNA you can read our article on RNA: RNA: Structure and Function

First, let me brief you on the RNA;

The RNA is ribonucleic acid; tRNA, rRNA and mRNA are three different types of it present in a cell. 

The tRNA is a transfer RNA that helps in transferring the information for protein synthesis, the rRNA is a ribosomal RNA located in the ribosome, reads the order of amino acids and the mRNA is a messenger RNA. The mRNA or messenger RNA has all the information for coding a particular protein. 

Later on, different levels of protein structure are formed.

Functionally, mRNA is transcribed from the DNA and translated into the protein via the translation pathway. The shRNA, miRNA and siRNA are other subsidiary RNA types present in a minor amount in a cell. Their main function is to regulate gene expression. The structure of some RNAs are shown here, 

The present article explains what siRNA is and how important it is! I will explain to you the importance of siRNA in the regulation of gene expression and its therapeutic applications.

Abbreviation  Full name 
mRNA  Messenger RNA 
miRNA  microRNA
shRNA  Small hairpin RNA 
siRNA  Small interfering RNA
RNAi  RNA interference
pri-miRNA Primary microRNA 
pre-miRNA  Precursor microRNA 
RISC RNA induced silencing complex 
Ago  Argonaute protein

So let’s start,

What is siRNA? 

In a layman, the siRNA is, we can the mRNA killer! meaning destroys “some of the mRNAs” to regulate the expression of genes.

The smaller double-stranded piece of RNA having a dinucleotide overhang at the 3’ end which functionally, degrade the mRNA and prevent the protein synthesis are siRNAs.

The siRNA is also known as small interfering ribonucleic acid or silencing RNA and is a molecule that prevents gene expression. This means it silence genes. The entire process of gene silencing through the siRNA is called a mechanism of RNA interference or siRNA knockdown.  

 The siRNA is functionally and structurally different from the other type of RNAs. Generally, other RNAs are single-stranded and made up of the long polynucleotide chain. On the other side, the siRNA is different. 

Structure of siRNA:

The siRNA is double-stranded, short and 20 to 25 nucleotides long. The source of siRNA is exogenous and functionally, it blocks protein translation. 

Apart from all these, one of the unique characters of the siRNA is the presence of the 3’ OH dinucleotide overhang. See the figure,

The structure of siRNA having a guided strand, passanger strand and the dinucleotide overhang at the 3 end.

The structure of siRNA having a guided strand, passenger strand and the dinucleotide overhang at the 3 ends.

Structurally, it is a dsRNA and shorter in length with an overhang at one end.

From the double-strand, one strand is known as a guided strand while the other is known as a passenger strandIt is also called a sense strand and antisense strand, respectively. In the year 1999, David Baulcombe and coworkers explained the role of the siRNA in the post-transcriptional modification. 

At a molecular level, it’s made up or Adenine, Uracil, Cytosine and Guanine. Two adjacent nucleotides are joined by the phosphodiester bond and two nucleotides of different strands are joined by hydrogen bonds. Dinucleotides on both the 3′ ends do not have hydrogen bonds.

Related article: What Is shRNA (Short-hairpin RNA)?  

Function of siRNA: 

The main function of siRNA is to protect the cell from exogenous mRNA attacks.

Functionally, the siRNA degrades the growing mRNA (exogenous as well as endogenous) and stops gene expression. The origin of the siRNA is exogeneous, it came from viral infections. 

The eukaryotic cells have a very good responsive defense system called RNA interference using with whole mechanism works.  

Now let us understand the entire mechanism in detail, 

RNA interference often denoted as RNAi is a biological process for mRNA degradation and subsequent gene silencing. In 1998, Fire and Mello unfold the mechanism of RNA interference. The role of siRNA in RNA interference was discovered in 1999. 

Once the retrovirus infects a cell it inserts its dsRNA into our cell. The specialized protein called ‘dicer’ having tetrameric manganese ions cuts or cleaves the dsRNA into smaller pieces. 

A special type of RNase, the dicer cleaves the RNA in a fashion that produces the dinucleotide overhang. These smaller fragments of dsRNA then incorporated into the protein complex having multiple subunits and form the RNAi-induced silencing complex, RISC

The RISC finds the appropriate mRNA target and cleaved it by a combination of endo and exonuclease activity. These smaller dsRNAs are ~22 to 25 basepair long and called as a small interfering RNA or siRNA. The siRNA also has the phosphate group at the 5’ end of it. 

It is believed that the overhang of dinucleotides is originated due to the activity of manganese ions present in the dicer. 

The guided strand of siRNA guides the protein complex to find the complementary dsRNA sequence present in a cell, once it is recognized, it is cleaved and destroyed. In this way, the natural RNA interference defense mechanism protects the cell from viral infection via the siRNA.

Similarly, it can also destroy our own mRNA by finding the complementary mRNA, henceforth, it modifies the properties of the chromosome by altering the epigenetic profile of the genome. 

Applications of siRNA: 

The present mechanism is actively present in almost all eukaryotes and works against viral infections. Nowadays, scientists are using this knowledge for gene silencing and regulating gene expression for therapeutic uses.

Scientists are now synthesizing the artificial siRNA molecules specific to the mRNA of a gene they wish to block. 

By using the viral vector based on nonviral-vector-based artificial methods of gene transfer, the siRNA can be inserted into the cell. Read more on viral vector-based and nonviral- vector-based gene delivery: Gene Therapy: Types, Vectors [Viral and Non-Viral], Process, Applications and Limitations.

This mechanism destroys the target mRNA and regulates protein synthesis.

Researchers are now trying to use the siRNA-mediated gene silencing method to suppress cancer-causing genes. 

The siRNA-mediated method is used in the gene knockout and gene knockdown methods for suppressing gene expression.

It is used in the target validation too, meaning, one can validate the target gene studied. 

It is also employed in pathway analysis and pathway identifications like cytokinesis, insulin signaling and cell defense mechanism, etc.

Further, It is applicable in gene redundancy and gene functional studies. 

The carbon-based and non-carbon-based nanoparticle-mediated siRNA therapy is used in the drug delivery to the brain.

Interestingly, in 2001, Elbashir et al used the synthetic siRNA molecule for artificial gene silencing. 

Challenges in the siRNA: 

The RNA interference by siRNA is a novel approach, researchers are not much aware of how to use it effectively. Numerous problems associated with the use of siRNA in therapeutics, some of those challenges are discussed here.

The nuclease present in the plasma and the tissue degrades the foreign siRNA oligo molecules, however, the siRNA via nanoparticles shown some promising results, as we stated in the above section. 

Further, the effect of the present therapy is fewer and tissue-specific thus it is restricted to the localized sites. 

Because of its larger size, it is very difficult to pass it through the cell membrane, although nano-vectors can transfer the siRNA effectively. 

Off targeting of the siRNA is one of the major challenges is the siRNA research as it also degrades other mRNAs too. 

It is very difficult to use the siRNA for therapeutic applications now, because of these challenges; notwithstanding, it will be applicable in the future. Using the expression vectors or bacterial vectors some of the problems can be solved but there are many other issues that scientists have to look after. 

Read more: siRNA vs miRNA: 10 Major Differences.

Example of therapeutic siRNA:

SM2181 AUCUGAAGAAGGAGAAAAATT UCCUUUCUUUCUUUCGAAUTT 2% mRNA inhibition 0.3 nM
SM2172 AUCUGAAGAAGGAGAAAAATT UUUUUCUCCUUCUUCAGAUTT 88 % target mRNA inhibition 0.3 nM

Now, these are the two examples of the siRNA with the sense strand and the antisense strand, one with the higher activity and one with the lowest activity. Also, the table shows the concentration of siRNA. 

The data for the siRNA is now available on siRNAmod.

Related articles:

  1. DNA story: The structure and function of DNA
  2. RNA: Structure and Function

Wrapping up:  

siRNA-mediated therapies are one of the most promising tools for the biopharmaceutical fields. The specificity is one of the major obstacles in recent days, albeit, it can be a therapeutic tool to cure disease. Cancer is the main target for researchers. The area isn’t much explored and that is why problems are many and results are less. 

For example, lentivirus-mediated RNA interferences give good results, are more effective and accurate but the use of retrovirus-like lentivirus is indeed dangerous. Ideally, it can’t be useful in human trials.

Though viral and non-viral vectors such as lipid-based, peptide-based, oligo-based and polymer-mediated delivery systems are now available for siRNA, every method has some limitations. 

If you are new to this article and do not understand several points, we have given links to study what is RNA interference, shRNA or miRNA, somewhere in this article. You can click it and read it.

Resources:

  1. Dana H, Chalbatani GM, Mahmoodzadeh H, et al. Molecular Mechanisms and Biological Functions of siRNA. Int J Biomed Sci. 2017;13(2):48–57.
  2. The molecule of the month; Small interfering RNA.