Kinetic selection of HPV 16 E6/E7-directed antisense nucleic acids: anti-proliferative effects on HPV 16-transformed...

The E6/E7-coding sequences of the human papillomavirus type 16 (HPV 16) were probed for kinetic accessibility in vitro by pools of catalytic antisense RNA. Only long-chain complementary RNA and very few antisense sequences with a 3' portion complementary to a 10 nt window within unspliced and spliced E6-coding target sequences showed fast annealing with k(ass) values of up to 10(4) M-1s-1 indicating that the majority of E6/E7 RNA sequences are unfavourable targets for antisense inhibitors and ribozymes. Fast-annealing antisense oligodeoxyribonucleotides directed against the window of 10 nt inhibited cell proliferation of HPV 16-transformed SiHa cells but not slow-annealing antisense species. Antisense RNA of several hundred nucleotides in length also showed significant anti-proliferative activity. Biological effects of antisense oligodeoxyribonucleotides were specific for the antisense sequence, could only be found in HPV-positive but not in HPV-negative cell lines, and were related to decreased levels of E7 protein and E6/E7-specific transcripts. This work suggests that HPV 16 E7/E6 sequences exhibit a low accessibility for antisense oligonucleotides. This can be overcome, however, by exploiting the relationship between fast annealing of antisense species and their increased efficacy in human cells.