Published online 23 July 2004
Nucleic Acids Research, Vol. 32 No. 13 © Oxford University Press 2004; all rights reserved
Sequence-specific artificial ribonucleases. I. Bis-imidazole-containing oligonucleotide conjugates prepared using precursor-based strategy
Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Sciences, Novosibirsk 630090, Russian Federation, 1 Wolfson Centre for Rational Design of Molecular Diagnostics, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PL, UK and 2 Fidelity Systems Inc., 7961 Cessna Avenue, Gaithersburg, MD 20879, USA
* To whom correspondence should be addressed. Tel: +7 3832 333761; Fax: +7 3832 333677; Email: marzen{at}niboch.nsc.ru
Received May 21, 2004; Revised and Accepted June 24, 2004
Antisense oligonucleotide conjugates, bearing constructs with two imidazole residues, were synthesized using a precursor-based technique employing post-synthetic histamine functionalization of oligonucleotides bearing methoxyoxalamido precursors at the 5'-termini. The conjugates were assessed in terms of their cleavage activities using both biochemical assays and conformational analysis by molecular modelling. The oligonucleotide part of the conjugates was complementary to the T-arm of yeast tRNAPhe (44–60 nt) and was expected to deliver imidazole groups near the fragile sequence C61-ACA-G65 of the tRNA. The conjugates showed ribonuclease activity at neutral pH and physiological temperature resulting in complete cleavage of the target RNA, mainly at the C63–A64 phosphodiester bond. For some constructs, cleavage was completed within 1–2 h under optimal conditions. Molecular modelling was used to determine the preferred orientation(s) of the cleaving group(s) in the complexes of the conjugates with RNA target. Cleaving constructs bearing two imidazole residues were found to be conformationally highly flexible, adopting no preferred specific conformation. No interactions other than complementary base pairing between the conjugates and the target were found to be the factors stabilizing the ‘active’ cleaving conformation(s).
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