ANTISENSE GENE INHIBITION BY C-5-SUBSTITUTED DEOXYURIDINE-CONTAINING OLIGODEOXYNUCLEOTIDES

Antisense oligodeoxynucleotides (ODNs) are capable of inhibiting gene expression via a RNase H mechanism in which the complementary RNA is d egraded by RNase H. C-5 propyne dU phosphorothioate ODNs bind selectiv ely and with high affinity to RNA within cells leading to potent antis ense inhibition of RNA translation. The effect that increasing steric bulk of C-5-substituted deoxyuridine analogs has on affinity for RNA a nd ability to inhibit gene expression is discussed. The relative bindi ng affinity was measured by thermal denaturation (Tm) analysis, and an tisense activity was determined by inhibition of SV40 T-antigen (TAg) expression in CV1 cells. The results show that antisense activity is n ot directly correlated to Tm measurements. In vitro analysis (RNase H cleavage, on-rates, and off-rates) and pre-formed ODN/RNA experiments indicate that RNase H activity and intracellular dissociation appear t o be major determinants of the antisense potency of the various substi tuted ODNs. The results of our analysis point to the unique ability of C-5 propyne dU ODNs to selectively bind to RNA within cells and activ ate cleavage of RNA by RNase H leading to potent inhibition of gene ex pression.