Selective inhibitory DNA aptamers of the human RNase H1

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)...

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Bibliographic Details
Published in:Nucleic acids research 2003-10, Vol.31 (19), p.5776-5788
Main Authors: Pileur, Frédéric, Andreola, Marie‐Line, Dausse, Eric, Michel, Justine, Moreau, Serge, Yamada, Hirofumi, Gaidamakov, Sergei A., Crouch, Robert J., Toulmé, Jean‐Jacques, Cazenave, Christian
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Directed Molecular Evolution
DNA - chemistry
DNA - pharmacology
DNA aptamers
Enzyme Inhibitors - chemistry
Enzyme Inhibitors - metabolism
Enzyme Inhibitors - pharmacology
Escherichia coli
G-Quadruplexes
Humans
Molecular Sequence Data
Nucleic Acid Conformation
Oligodeoxyribonucleotides - chemistry
Oligodeoxyribonucleotides - metabolism
Oligodeoxyribonucleotides - pharmacology
Oligonucleotides, Antisense - pharmacology
Protein Biosynthesis
Rabbits
Reticulocytes - metabolism
Ribonuclease H - antagonists & inhibitors
Ribonuclease H - metabolism
ribonuclease H1
Sequence Analysis, DNA
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Summary: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.
ISSN:0305-1048
1362-4962
1362-4962
DOI:10.1093/nar/gkg748