We describe our initial application of a biochemical strategy, comprising combinatorial screening and rational optimization, which directly identifies oligonucleotides with maximum affinity (per unit length), specificity, and rates of hybridization to structurally preferred sites on folded RNA, to the problem of design of antisense oligonucleotides active against the hepatitis C virus (HCV). A fully randomized sequence DNA oligonucleotide (10-mer) library was equilibrated with each of two folded RNA fragments (200 and 370 nucleotides (nt)), together spanning the 5' 440 nt of an HCV transcript (by overlapping 130 nt), which were varied over a range of concentrations. The equilibrations were performed in solution under conditions determined to preserve RNA structure and to limit all RNA-DNA library oligonucleotide interactions to 1:1 stoichiometry. Subsequent Escherichia coli RNase H (endoribonuclease H: EC 3.1.26.4) cleavage analysis identified two preferred sites of highest affinity heteroduplex hybridization. The lengths and sequences of different substitute chemistry oligonucleotides complementary to these sites were rationally optimized using an iterative and quantitative analysis of binding affinity and specificity. Thus, DNA oligonucleotides that hybridized with the same affinity to the preferred sites in the folded RNA fragments found by screening as to short (< or = 25 nt) RNA complements were identified but were found to vary in length (10-18 nt) from site to site. Phosphorothioate (P=S) and 2'-fluoro (2'-F) uniformly substituted oligonucleotides also were found, which hybridized optimally to these sites, supporting the design of short (10-15-nt) and maximally specific oligonucleotides that are more nuclease-resistant (via P=S) and have higher affinity (via 2'-F) than DNA. Finally, the affinities of DNA and uniform 2'-F-, P=S-substituted 10-20-mer oligonucleotide complements for the best hybridization site, from HCV nt 355 to nt 364-374, closely corresponded to antisense mechanism inhibition activities in an in vitro translation assay and in a human cell-based HCV core protein expression assay, respectively. These results validate our strategy for the selection of hybridization-optimized and biologically active antisense oligonucleotides targeting HCV RNA and support the potential for utility in further applications.