In vitro selection of fast-hybridizing and effective antisense RNAs directed against the human immunodeficiency virus...

The rate of double strand formation between procaryotic antisense RNA and complementary RNA in vitro is known to correlate with the effectiveness of antisense RNA in vivo. In this work, an in vitro assay for determining the hybridization rates of a large number of antisense RNA species was developed. A set of HIV-1-directed antisense RNAs with the same 5'-end but successively shortened 3'-ends was produced by alkaline hydrolysis of a 150 nt HIV-1-directed antisense transcript. This mixture was used to determine hybridization rates for individual chain lengths with a complementary HIV-1-derived RNA in vitro. The second order binding rate constants of individual antisense RNA species differed by more than a factor of 100, although in some cases, slow-hybridizing and fast-hybridizing antisense RNAs differed by only two or three 3'-terminally-located nucleotides. The results indicated that there was not a trivial dependence of binding rates on the chain length of antisense RNAs. Further, the binding rate constants determined in vitro for individual antisense RNA species correlated with the extent of inhibition of HIV-1 replication in vivo.