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(Received for publication, April 15, 1996, and in revised form, August 27, 1996)
From the ¶ Department of Research Medicinal Chemistry, the
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
Volume 272, Number 1,
Issue of January 3, 1997
pp. 626-638
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
,¶
Department of Molecular, Cellular, and Structural
Biology, and the § Department of Infectious Diseases, Isis
Pharmaceuticals, Carlsbad, California 92008
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 (
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.
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