Jc. Francois et al., RECOGNITION AND CLEAVAGE OF HAIRPIN STRUCTURES IN NUCLEIC-ACIDS BY OLIGODEOXYNUCLEOTIDES, Nucleic acids research, 22(19), 1994, pp. 3943-3950
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.