Antisense/ASO & siRNA Applications

Antisense/ASO & siRNA Applications

When designing an antisense oligo, both the stability of the duplex formed with the mRNA, and the inherent stability of the antisense oligo itself (as measured by its cellular half-life), are crucial to successful inhibition. Vigorous research activity has been devoted in developing novel base analogs and backbone linkages capable of making an antisense oligo resistant to nuclease degradation and making it have strong hybridization properties. Examples include the classical phosphorothiolate linkages, propyne base analogs, and 2'-OMethyl bases. Substitution of a phosphorothiolate linkage for a phosphodiester linkage in an oligo renders that position resistant to nuclease degradation. However, it also lowers the Tm of any duplex formed by such an oligo by about 0.5C per linkage. Thus, to strike a proper balance between increased nuclease resistance and decreased duplex stability, researchers often limit phosphorothiolation to the three bases on each of the 5'- and 3'-ends (end caps) to minimize exonuclease degradation. Phosphorothiolation does not affect RNase H activity. Addition of a propyne base analog to phosphorothiolated oligo sometimes can compensate for the lowered Tm and improve duplex stability without affecting nuclease resistance (3). As an alternative to phosphorothiolation, 2'-O Methyl RNA bases can be incorporated at some or all of the positions of an antisense oligo. The resulting oligo is both resistant to nuclease degradation at the modified positions and has higher duplex stability (higher Tm) than its unmodified counterpart. However, 2'-O Methyl antisense oligos do not activate RNase H activity, but they can suppress gene expression by blocking the mRNA translation process via steric hindrance (4). However, antisense oligos having a central core of several natural (2'-OH) DNA bases and 2'-O Methyl-substituted bases on the ends (gapmers or gap chimeras) form duplexes with mRNA that are degraded by RNase H (5).

References

(1) Sazani, P., Kole, R. Therapeutic potential of antisense oligonucleotides as modulators of alternative splicing. J. Clin. Invest. (2003), 112: 481-486.
(2) Juliano, R., Alam, Md.R., Dixit, V., Kang, H. Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides. Nucleic Acids Res. (2008), 36: 4158-4171.
(3) Chan, J.H., Lim, S., Wong, W.S. Antisense oligonucleotides: from design to therapeutic applications. Clin. Exp. Pharmacol. Physiol. (2006), 33: 533-540.
(4) Kurreck, J. Antisense technologies. Improvement through novel chemical modifications. Eur. J. Biochem. (2003), 270: 1628-1644.
(5) Crooke, S.T. Progress in antisense technology. Annu. Rev. Med. (2004), 55: 61-95.