Recognition and cleavage of hairpin structures in nucleic acids by oligodeoxynucleotides.
The possibility of designing antisense oligodeoxynucleotides complementary to non-adjacent single-stranded sequences containing hairpin structures was studied using a DNA model system. The structure and stability of complexes formed by a 17mer oligonucleotide with DNA fragments containing hairpin structures was investigated by spectroscopic measurements (melting curves) and chemical reactions (osmium tetroxide reaction, copper-phenanthroline cleavage). A three-way junction was formed when the oligonucleotide was bound to both sides of the hairpin structure. When the complementary sequences of the two parts of the oligonucleotide were separated by a sequence which could not form a hairpin, the oligonucleotide exhibited a slightly weaker binding than to the hairpin-containing target. An oligodeoxynucleotide-phenanthroline conjugate was designed to form Watson-Crick base pairs with two single-stranded regions flanking a hairpin structure in a DNA fragment. In the presence of Cu2+ ions and a reducing agent, two main cleavage sites were observed at the end of the duplex structure formed by the oligonucleotide-phenanthroline conjugate with its target sequence. Competition experiments showed that both parts of the oligonucleotide must be bound in order to observe sequence-specific cleavage. Cleavage was still observed with target sequences which could not form a hairpin, provided the reaction was carried out at lower temperatures. These results show that sequence-specific recognition and modification (cleavage) can be achieved with antisense oligonucleotides which bind to non-adjacent sequences in a single-stranded nucleic acid.
François
J C
JC
Laboratoire de Biophysique, INSERM U.201, CNRS U.A. 481, Paris.
Thuong
N T
NT
Hélène
C
C
D001482:Base Composition; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D007364:Intercalating Agents; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism*; D016376:Oligonucleotides, Antisense; D009993:Osmium Tetroxide; D010618:Phenanthrolines / Q000378:metabolism; D013053:Spectrophotometry
D007364:Intercalating Agents; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D010618:Phenanthrolines; D004247:DNA; D009993:Osmium Tetroxide; C025205:1,10-phenanthroline