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The following points highlight the two main types of antisense methods that can be employed to inhibit the expression of a target gene. These antisense methods are antisense oligonucleotides and antisense RNA. All these methods are basically designed to blockade or degradation of a specific target mRNA as a consequence of binding of nucleic acids complementary to sequence of the mRNA.
Method # 1. Antisense Oligonucleotides (AON):
Antisense oligonucleotides (AON) method involves direct supply of single-stranded ODN of 15-25 nucleotides, which binds to a specific mRNA. As a consequence of DNA-RNA duplex formation via Watson-Crick base pairing, translation of specific mRNA into corresponding protein is blocked, its degradation or altering translation. Degradation is achieved by recruiting enzyme RNase H and translation is arrested by altering at ribosome binding sites in mRNA.
Antisense oligonucleotide molecules can be employed in cell culture, and these oligonucleotides can suppress gene expression in limited extent due to inefficient delivery to the cells. Several ODN delivery systems have been developed in order to increase stability under physiological conditions.
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Several examples of delivery systems or vectors which can carry naked DNA effectively into cells include polyethylene glycol (PEG), conjugated liposome and alginate or poly-L-lysine micro-particles. It is, however, due to inconsistent and unsatisfactory delivery system, there is a requirement of more efficient, controllable delivery system is warranted.
Several strategies have been enforced to increase the antisense activity and stability of ODN molecule. Chemical modification of antisense oligonucleotide proved satisfactory results. Towards these directions various modifications of sugar, base and phosphate backbone AON’s have been attempted. Chemical modifications provide enhanced resistance to various cellular nucleases after AON Molecules enters the cell.
Some of the chemical modifications include lethering of various chromophores like pyrene, anthracene to 3′ and/or 5′ end of antisense oligonucleotides resulted in enhanced binding affinity to the target, stability towards nuclease and efficient recruitment of RNase H in a faster rate of degradation. Another modified AON, a chimeric locked nucleic acid DNA copolymer exhibit potential and specific action including recruitment of RNase H.
Antisense oligonucleotide once placed inside the cell, its next destiny is to reach the site of mRNA and able to bind to its mRNA target. The target mRNA generally shows inaccessible to binding of AON due to its intramolecular base pairs formed between complementary sequences in single stranded mRNA. Thus, antisense oligonucletide methods of gene silencing still shows limited success and require many refined strategies.
Method # 2. Antisense RNA:
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Another gene silencing approach is the antisense RNA technique. The technique involves the introduction of expression vector contains antisense gene into host cells, which inhibits expression of the gene from which it is derived.
It is presumed that expression of antisense gene results in RNA duplex forms between antisense RNA and transcript complementary mRNA preventing translation, thereby preventing expression of specific target gene.
