RECOGNITION AND CLEAVAGE OF HAIRPIN STRUCTURES IN NUCLEIC-ACIDS BY OLIGODEOXYNUCLEOTIDES

The possibility of designing antisense oligodeoxynucleotides complemen tary to non-adjacent single-stranded sequences containing hairpin stru ctures was studied using a DNA model system. The structure and stabili ty of complexes formed by a 17mer oligonucleotide with DNA fragments c ontaining hairpin structures was investigated by spectroscopic measure ments (melting curves) and chemical reactions (osmium tetroxide reacti on, copper-phenanthrotine cleavage). A three-way junction was formed w hen the oligonucleotide was bound to both sides of the hairpin structu re. When the complementary sequences of the two parts of the oligonucl eotide were separated by a sequence which could not form a hairpin, th e oligonucleotide exhibited a slightly weaker binding than to the hair pin-containing target. An oligodeoxynucleotide-phenanthroline conjugat e was designed to form Watson-Crick base pairs with two single-strande d regions flanking a hairpin structure in a DNA fragment. In the prese nce of Cu2+ ions and a reducing agent, two main cleavage sites were ob served at the end of the duplex structure formed by the oligonucleotid e - phenanthroline conjugate with its target sequence. Competition exp eriments showed that both parts of the oligonucleotide must be bound i n order to observe sequence-specific cleavage. Cleavage was still obse rved with target sequences which could not form a hairpin, provided th e reaction was carried out at lower temperatures. These results show t hat sequence-specific recognition and modification (cleavage) can be a chieved with antisense oligonucleotides which bind to non-adjacent seq uences in a single-stranded nucleic acid.