Nuclease Resistance Applications

Nuclease Resistance Applications

For antisense or RNAi applications, incorporation of modifications conferring nuclease resistance is essentially indispensable and such modifications are used intensely. The most popular modification used for this purpose is phosphorothiolation, in which a phosphodiester backbone linkage is replaced by a phosphothiolate linkage. Such linkages are highly resistant to nuclease degradation, but they also lower duplex stability by about 0.5C per phosphorothioate linkage. However, judicious use of this modification (for example, placing them only at the three endmost bases of each end of the oligo to minimize exonuclease degradation) can produce excellent nuclease resistance while still maintaining reasonably good duplex stability (2). 2'-O Methyl (or other 2'-O-substituted) RNA bases also confer nuclease resistance to an oligo, and have the added benefit of increasing duplex stability as well (3,4). However, duplexes formed between oligos having 2'-OMethyl bases at all positions and RNA are incapable of activating RNase H activity (3), and this fact must be kept in mind if the user wishes to use such oligos for antisense applications. Recently, "mirror-image" (L)-nucleotide base phosphoramidites have become available as well. Oligonucleotides containing (L)-nucleotides are completely immune to nuclease attack at the incorporated positions (5). While (L)-nucleotides also do not base pair with natural (D)-nucleotides (6), they still could potentially be incorporated in, for example, the stems of molecular beacons to protect them from degradation by exonucleases.

References

(1) Cazenave, C., Chevrier, M., Nguyen, T.T., Helene, C. Rate of degradation of [alpha]- and [beta]-oligodeoxynucleotides in Xenopus oocytes. Implications for anti-messenger strategies. Nucleic Acids Res. (1987), 15: 10507-10521.
(2) Pandolfi, D., Rauzi, F., Capobianco, M.L. Evaluation of different types of end-capping modifications on the stability of oligonucleotides toward 3'- and 5'-exonucleases. Nucleosides Nucleotides (1999), 18: 2051-2069.
(3) Monia, B.P., Lesnik, E.A., Gonzalez, C., Lima, W.F., McGee, D., Guinosso, C.J., Kawasaki, A.M., Cook, P.D., Frier, S.M. Evaluation of 2'-Modified Oligonucleotides Containing 2'-Deoxy Gaps as Antisense Inhibitors of Gene Expression. J. Biol. Chem. (1993), 268: 14514-14522.
(4) Monia, B.P., Johnston, J.F., Sasmor, H., Cummins, L.L. Nuclease Resistance and Antisense Activity of Modified Oligonucleotides Targeted to Ha-ras. J. Biol. Chem. (1996), 271: 14533-14540.
(5) Sooter, L.J., Ellington, A.D. Reflections on a Novel Therapeutic Candidate. Chem. & Biol. (2002), 9: 857-858.
(6) Urata, H., Ogura, E., Shinohara, K., Ueda, Y., Akagi, M. Synthesis and properties of mirror-image DNA. Nucleic Acids Res. (1992), 20: 3325-3332.
(7) Eder, P.S., DeVine, R.J., Dagle, J.M., Walder, J.A. Substrate specificity and kinetics of degradation of antisense oligonucleotides by a 3' exonuclease in plasma, Antisense Res. Dev. (1991), 1: 141-151.
(8) Dagel, J.M., Weeks, D.L., Walder, J.A. Pathways of degradation and mechanism of action of antisense oligonucleotides in Xenopus laevis embryos. Antisense Res. Dev. (1991), 1: 11-20.