Basis of the technology
A sense strand is a 5â€² to 3â€² mRNA molecule or DNA molecule. The complementary strand to this strand is called an antisense. The antisense technology works by binding of antisense strand with the targeted sense strand through hydrogen bond. The double stranded molecule will be recognized as a foreign molecule and is susceptible to degradation.
Though, as the DNA is double helix, it can be used in the antisense technology; resulting in the formation of triplex. Antisense strand can be:-
1) DNA: - A DNA antisense strand is of approximately 17 bases long.
2) RNA (either catalytic or non catalytic):- 13 base long RNA antisense strand functions properly. The catalytic antisense strands are called ribozymes, which cuts the RNA molecules at specific position while the non catalytic antisense strand blocks the processing of RNA. More widely used than DNA
Stages of inhibition of antisense
Antisense strand can act at three stages in cells
1) Transcription is the first stage at which a target gene can be blocked. But there is no proper evidence for this.
2) Second stage is the processing of the RNA. When antisense strand binds with the sense strand, there is a formation of duplex. RNA-RNA duplex is unstable and is susceptible to nucleases, which quickly degrade the former. But there is no direct evidence for duplex formation perhaps due to quick disintegration of double stranded RNA (mRNA-antisense hybrid), thus making the sense or target mRNA unavailable for translation.
3) Translation is the third stage of inhibition. Antisense strand competes with the ribosomes for the binding site of sense RNA, thus inhibiting the translation. Once the ribosome binds the mRNA, it will translate to form functional protein. So, for the significant inhibition of target mRNA, antisense strand must bind to it before ribosome.
Antisense RNA is constructed by reversing the orientation of a gene with regard to its promoter so that the antisense strand is transcribed e.g., an antisense thymidine kinase gene inhibits synthesis of thymidine kinase from the endogenous gene. When an antisense strand binds to the targeted sense strand, double helix forms and cell will recognize this double helix as a foreign molecule and work in a direction to destroy it. Binding of antisense strand to their target sense strand activates a cellular enzyme called RNase H. This enzyme degrades the target RNA, thus inhibits the formation of a particular protein.
On the basis of mechanism of regulation of antisense RNA, they are of 3 types:
Class I: - antisense RNA are directly complementary to the coding regions of the target mRNA. Thus binding of antisense RNA to the target mRNA results in its destabilization or directly inhibits the translation.
Class II: - these antisense RNA binds to the non-coding regions of the target RNA resulting in indirect effects i.e. form alternate secondary structure that sequesters the ribosome binding sites.
Class III: - antisense RNA regulate transcription of target mRNA by mechanism similar to transcriptional attenuation.
Applications of antisense technology
1) Artificial antisense RNA regulation of gene expression
Antisense RNA has also been used to artificially modulate gene expression in plants and animals. To determine the optimum requirement for an efficient antisense RNA regulation, varied lengths as well as regions of sense gene of B-galactosidase were targeted. This experiment leads to the following conclusions:-
• The antisense RNA must be complementary to the 5'end of sense mRNA and a functional ribosome binding site on the 5' end.
• The molar ratio between antisense and sense RNA varies from 60:1 to 600:1.
• In exceptional cases the molar ratio may be 1:1.
2) Application in crop improvement: - The most important example of antisense technology is in crop improvement regarding the development of Flavr Savr tomato. This involves the inhibition of polygalacturonase (PG) gene, which codes for the polygalacturonase enzyme. This enzyme causes the degradation of cell wall that result in fruit ripening. Inhibition of PG gene expression greatly reduces the fruit ripening, thus improves shelf life of tomatoes. Antisense technology has also been applied for potato crop to reduce the discoloration of tubers after bruising.
3) Virus resistance by antisense RNA: - this approach has also applied to reduce the accumulation of viruses in plants. Viruses against which this technology showed good results include potato virus X (PVX), tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV)
4) Antisense technology in the treatment of genetic disorders
Antisense technology has also found implications to tackle the effects of genetic disorders and infections. Diseases are often characterized by insufficient or excessive production of proteins, antisense therapy can halt the production of these protein products, thereby curing the disease.
In conclusion, antisense technology offers almost unlimited scope for the development of new methods of drug design. It is one of the most approved approaches among several others, for inactivating a single chosen gene. However, the full commitment of this promise is yet to be established.
About Author / Additional Info:
Deepak Harishchandra WaghmarePune, Maharashtra, India
I am a Project fellow on DST-SERB funded project "Phytochemical Potential of medicinal plants as antisickling agents in management of sickle cell disease"
Shanky BhatNew Delhi, Delhi
AMAN. GUPTAnew delhi, new delhi
i am the student of b-tech biotech in jaypee institute of information technology and i have done training in AIIMS on cancer IRCH department.and i want to become a great scientist in biotech field.
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