GENE SILENCING ANTISENSE RNA & siRNA TECHNOLOGIES

Gene silencing is a general term describing epigenetic (study of heritable changes in phenotype (appearance) or gene expression caused by mechanisms other than changes in the underlying DNA sequence), processes of gene regulation The term gene silencing is generally used to describe the "switching off" of a gene by a mechanism other than genetic modification i.e, a gene which would be expressed (turned on) under normal circumstances is switched off by machinery in the cell

Genes

are

regulated
or

at

either

the

transcriptional level.

post-transcriptional

Transcriptional gene silencing is the result of histone modifications, creating an environment of heterochromatin around a gene that makes it inaccessible to transcriptional machinery (RNA polymerase, transcription factors, etc.) Post-transcriptional gene silencing is the result of mRNA of a particular gene being destroyed or blocked The destruction of the mRNA prevents translation to form an active gene product (in most cases, a protein) A common mechanism of post-transcriptional gene silencing is RNAi Both transcriptional and post-transcriptional gene silencing are used to regulate endogenous genes

Mechanisms of gene silencing also protect the organism's genome from transposons and viruses Gene silencing is thus a part of an ancient immune system protecting from such infectious DNA elements

siRNA
Small interfering RNA (siRNA), or short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 nucleotides in length Play a variety of roles in biology

The most notable role - involvement in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene
In addition to its role in the RNAi pathway, siRNA also acts in RNAi-related pathways, e.g., as an antiviral mechanism or in shaping the chromatin structure of a genome

siRNA Pathway ds RNA is introduced into the cell either using a short oligo (Short Interfering RNA) siRNA or a DNA plasmid from which an siRNA can be transcribed The Dicer protein in the cell digests dsRNA into 21 bp ds RNA (siRNA) siRNAs are integrated into the RNA Induced Silencing Complex (RISC) Within this RISC complex, the double stranded siRNAs undergo strand separation.

The antisense strand hybridizes to the complementary / target mRNA in the cell Nucleases within the degrade targeted mRNA activated RISC

The fragmented mRNA translated into protein

cannot

be

This means the protein cannot be expressed, resulting in knockout of the protein.

Messenger RNA (mRNA) is a single stranded molecule that is used as the template for protein translation It is possible for RNA to form duplexes, similar to DNA, with a second sequence of RNA complementary to the first strand This second sequence is called antisense RNA (Figure) The formation of double stranded RNA can inhibit gene expression in many different organisms including plants, flies, worms and fungi.
Formation of antisense RNA blocks translation.

Co-Suppression The first discovery of this type of gene inhibition in plants occurred in petunias Research was carried out to deepen the purple color of the flowers by injecting the gene responsible into the petunias Instead of a darker flower, the petunias were either spotted or completely white A variegated petunia. Upon injection of the gene responsible for purple colouring in petunias, the flowers became variegated or white rather than deeper purple as was expected

This phenomenon is termed co-suppression, since both the expression of the existing gene (the initial purple colour), and the introduced gene (to deepen the purple) were suppressed Co-suppression has since been found in many other plant species and also in fungi It is now known that double stranded RNA is responsible for this effect.

aRNA (antisense interference)

RNA)

and

RNAi

(RNA

When antisense RNA (aRNA) is introduced into a cell, it binds to the already present sense RNA to inhibit gene expression.

What would happen if sense RNA is prepared and introduced into the cell? Since two strands of sense RNA do not bind to each other, it is thought that nothing would happen with additional sense RNA, But the opposite happens! The new sense RNA suppresses gene expression similar to aRNA The reason is that preparations of sense RNA actually contain contaminating strands of antisense RNA The sense and antisense strands bind to each other, forming a helix.

This double helix is the actual suppressor of its corresponding gene The suppression of a gene by its corresponding double stranded RNA is called RNA interference (RNAi), or posttranscriptional gene silencing (PTGS). RNA double helix, is also formed by the sense RNA of the cell and the introduced antisense RNA which also leads to gene suppression How does the double stranded RNA cause gene suppression?

Since the only RNA found in a cell should be single stranded, the presence of double stranded RNA signals is an abnormality

The cell has a specific enzyme (in Drosophila it is called Dicer) that recognizes the double stranded RNA and chops it up into small fragments between 21-25 base pairs in length These short RNA fragments are called small interfering RNA, or siRNA These siRNA bind to the RNAinduced silencing complex (RISC) The RISC is activated when the siRNA unwinds and the activated complex binds to the corresponding mRNA using the antisense RNA

The RISC contains an enzyme to cleave the bound mRNA (called Slicer in Drosophila) and therefore cause gene suppression Once the mRNA has been cleaved, it can no longer be translated into functional protein Application of antisense RNA A gene encoding the antisense RNA can be introduced fairly easily into organisms by using a plasmid vector or using a gene gun that shoots microscopic tungsten pellets coated with the gene into the plant cells

Once the antisense RNA is introduced, it will specifically inhibit the synthesis of the target protein by binding to mRNA

This is a quick way to create a knockout organism to study gene function Using antisense RNA and suppression of protein synthesis is an exciting prospect and finds varied applications 1. Antisense RNA is being investigated for use in cancer therapy Injecting aRNA that is complementary to the proto-oncogene BCL-2 may be useful for treating some B-cell lymphomas and leukemias Antisense oligodeoxynucleotides (ODNs) are also being studied for human therapy.

ODNs are similar to antisense RNA, but they are made synthetically and are deoxynucleotides (like those in DNA) rather than nucleotides ODNs are being tested for their effectiveness against HIV-1, cytomegalovirus (a member of the herpesvirus group), asthma and certain cancers. 2. Antisense RNA methods have also been used for commercial food production e.g., Flavr Savr tomato - a genetically modified tomato This tomato was developed by Calgene Inc. of Davis, California in 1991 and was approved by the U.S. FDA in 1994

was the first commercially grown genetically engineered food to be granted a license for human consumption One of the problems associated with tomato farming is that the fruit must be picked while still green in order to be shipped to market without being crushed The enzyme that causes softening in tomatoes is polygalacturonase (PG) This enzyme breaks down pectin in the cell wall as the tomato ripens, leading to a softer fruit Calgene (a biotech company in California) suppressed the expression of the gene encoding PG by introducing a gene encoding the antisense strand of the mRNA.

When the introduced gene was expressed, the antisense strand bound to the PG mRNA, suppressed the translation of the enzyme The Flavr Savr tomatoes therefore had low PG levels and remained firmer when ripe Hence, the Flavr Savr tomatoes can ripen on the vine and then be shipped to market.