Nucleic Acids Research, 2003, Vol. 31, No. 19 5776-5788
© 2003 Oxford University Press
Selective inhibitory DNA aptamers of the human RNase H1
1 INSERM U386, IFR Pathologies Infectieuses, Université Victor Segalen Bordeaux 2, 146, rue Léo Saignat, 33076 Bordeaux cedex, France, 2 UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146, rue Léo Saignat, 33076 Bordeaux cedex, France, 3 Institut Européen de Chimie et Biologie, CNRS FRE 2247, 33402 Talence, France and 4 Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institute of Health, 6 Center Drive MSC 2790, 9000 Rockville Pike, Building 6B, Room 2B-231, Bethesda, MD 20892-2790, USA
*To whom correspondence should be addressed. Tel: +33 5 57 57 10 14; Fax: +33 5 57 57 10 15; Email: christian.cazenave{at}bordeaux.inserm.fr
Present address:
Frédéric Pileur, Max-Planck-Institut für Molekulare Physiologie, Otto Hahn Strasse 11, 44227 Dortmund, Germany
This paper is dedicated to the memory of Prof. Claude Hélène
Human RNase H1 binds double-stranded RNA via its N-terminal domain and RNA–DNA hybrid via its C-terminal RNase H domain, the latter being closely related to Escherichia coli RNase HI. Using SELEX, we have generated a set of DNA sequences that can bind efficiently (Kd values ranging from 10 to 80 nM) to the human RNase H1. None of them could fold into a simple perfect double-stranded DNA hairpin confirming that double-stranded DNA does not constitute a trivial ligand for the enzyme. Only two of the 37 DNA aptamers selected were inhibitors of human RNase H1 activity. The two inhibitory oligomers, V-2 and VI-2, were quite different in structure with V-2 folding into a large, imperfect but stable hairpin loop. The VI-2 structure consists of a central region unimolecular quadruplex formed by stacking of two guanine quartets flanked by the 5' and 3' tails that form a stem of six base pairs. Base pairing between the 5' and 3' tails appears crucial for conferring the inhibitory properties to the aptamer. Finally, the inhibitory aptamers were capable of completely abolishing the action of an antisense oligonucleotide in a rabbit reticulocyte lysate supplemented with human RNase H1, with IC50 ranging from 50 to 100 nM.
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