| Abstract
| - The origins of the substrate specificity of Escherichia coli RNase H1 (termed RNase H here),an enzyme that hydrolyzes the RNA strand of DNA−RNA hybrids, are not understood at present. Althoughthe enzyme binds double-stranded RNA, no cleavage occurs with such duplexes [Lima, W. F., and Crooke,S. T. (1997) Biochemistry 36, 390]. Therefore, the hybrid substrates may not adopt a canonical A-formgeometry. Furthermore, RNase H is exquisitely sensitive to chemical modification of the DNA strands inhybrid duplexes. This is particularly relevant to the RNase H-dependent pathway of antisense action.Thus, only very few of the modifications currently being evaluated as antisense therapeutics are toleratedby the enzyme, among them phosphorothioate DNA (PS-DNA). Recently, hybrids of RNA andarabinonucleic acid (ANA) as well as the 2‘F-ANA analogue were shown to be substrates of RNase H[Damha, M. J., et al. (1998) J. Am. Chem. Soc.120, 12976]. Using X-ray crystallography, we demonstratehere that ANA analogues, such as 2‘F-ANA [Berger, I., et al. (1998) Nucleic Acids Res.26, 2473] and[3.3.0]bicyclo-ANA (bc-ANA), may not be able to adopt sugar puckers that are compatible with pure A-or a B-form duplex geometries, but rather prefer the intermediate O4‘-endo conformation. On the basis ofthe observed conformations of these ANA analogues in a DNA dodecamer duplex, we have modeled aduplex of an all-C3‘-endo RNA strand and an all-O4‘-endo 2‘F-ANA strand. This duplex exhibits a minorgroove width that is intermediate between that of A-form RNA and B-form DNA, a feature that may beexploited by the enzyme in differentiating between RNA duplexes and DNA−RNA hybrids. Therefore,the combination of the established structural and functional properties of ANA analogues helps settleexisting controversies concerning the discrimination of substrates by RNase H. Knowlegde of the structureof an analogue that exhibits enhanced RNA affinity while not interfering with RNase H activity mayprove helpful in the design of future antisense modifications.
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