Specificity of antisense oligonucleotides

Abstract

Antisense oligodeoxynucleotides that are sufficiently long to specify unique species of mRNA may direct ribonuclease H (RNase H) to cleave nontargeted mRNAs at sites of partial complementarity, both in cell-free systems and in living cells. Specificity of antisense action against selected gene expression may be achieved by increasing the stringency of hybridization under constant physiological conditions through incorporation of a limited number of helix-destabilizing methylphosphonate analogue backbone modifications in the molecules. Chimeric antisense oligodeoxynucleotides with terminal, nonionic methylphosphonodiester linkages were protected from exonuclease degradation, and exhibited reduced sensitivity to endonuclease attack relative to unmodified oligodeoxynucleotides. The reduced hybridization potential of the chimeric oligomers correlated with increased RNase H-mediated antisense activity at the target site in cell-free systems, and reduced rates of cleavage at sites of partial complementarity. Using this strategy, single base discrimination was achieved for destruction of p53 mRNA in living human leukemia cells.