Nuclease Resistance Design and Protocols

Nuclease Resistance Design / Protocol

While oligonucleotides are quickly degraded (typically within 15-30 minutes) by nucleases in both in vitro and in vivo contexts, the need to incorporate nuclease resistance into oligonucleotides is critically important for in vivo applications. Within serum or the cell, oligonucleotides can be degraded by both endo- and exonucleases. In serum, the 3'-exonucleases are of greatest concern (7), while within the cell, both 3'- and 5'-exonucleases are problematic (8). Endonucleases can also be an issue in those cases where the oligo contains a restriction site.

Based on the above, designing a nuclease-resistant oligonucleotide for in vivo applications primarily involves modifying it so as to protect it from exonucleases, while minimizing potential deleterious side-effects (such as reduced duplex stability, increased toxicity, or induction of off-target biological effects). The simplest and most cost-effective way to do this is to design the oligo as a 'gapmer', in which the linkages of the three end-most 5'- and 3'-bases are phosphorothiolated, with the remaining bases in the middle having native phosphorodiester linkages. Such phosphorothiolated 'gapmers' are highly resistant to both 5'- and 3'-exonuclease degradation. In addition, because phosphorothiolation lowers the binding affinity of the oligo for its target (Tm of the oligo-target duplex is lowered between 0.5C and 1.5C per linkage, depending on sequence), use of only six such linkages often yields an acceptable balance between nuclease resistance and binding affinity (if increased binding affinity is required, other modifications can also be incorporated into the oligo, such as 2'-fluoro pyrimidines, 2'-O-methyl RNA bases, or C5-propyne pyrimidines). Finally, since large numbers of phosphorothiolate linkages can be toxic (due to the presence of sulfur), using only a small number of such linkages in an oligo minimizes cellular toxicity.

If phosphorothiolation is not desired, other modifications can be used. One option is to use methylphosphonates instead of phosphorothiolation for the 5'- and 3'-end positions of the 'gapmer'. Methylphosphonates lower an oligo's binding affinity more than phosphorothiolation, however, so the use of additional modifications, such as 2'-fluoro-pyrimidines, is advisable to counteract this effect. More commonly, the substitution of 2'-O-methyl RNA bases at some or all positions of an oligo is used as an alternative to phosphorothiolation. Since the nuclease resistance conferred by 2'-O-methyl RNA lies between that of standard bases (no resistance) and phosphorothiolation (highly resistant), extensive/complete 2'-O-methylation is frequently chosen when a high level of nuclease resistance is required. 2'-O-methylation also confers higher binding affinity (that is, higher duplex Tm) to the oligo for its target, a desirable property, in many cases.

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.