Stable oligonucleotide-directed triplex formation at target sites with CG interruptions: strong sequence-specific recognition by 2',4'-bridged nucleic-acid-containing 2-pyridones under physiological conditions.

Abstract

A sequence of double-stranded DNA (dsDNA) which can be recognized by a triplex-forming oligonucleotide (TFO) is limited to a homopurine-homopyrimidine sequence. To develop novel nucleoside analogues which recognize CG interruption in homopurine-homopyrimidine dsDNA, we synthesized a novel 2'-O,4'-C-methyleneribonucleic acid (2'-O,4'-C-methylene bridged nucleic acid; 2',4'-BNA) that bears the unnatural nucleobases, 2-pyridone (PB) or its 5-methyl congener (mPB); these analogues were introduced into pyrimidine TFOs using a DNA synthesizer. A TFO with a 2'-deoxy-beta-D-ribofuranosyl-2-pyridone (P) or 2',4'-BNA abasic monomer (HB) was also synthesized. The triplex-forming ability of various synthesized 15-mer TFOs and the corresponding homopurine-homopyrimidine dsDNA, which contained a single pyrimidine-purine (PyPu) interruption, was examined in UV melting experiments. It was found that PB and mPB in the TFOs successfully recognized CG interruption under physiological conditions (7 mM sodium phosphate, 140 mM KCl, 5 mM spermine, pH 7.0). Furthermore, triplex formation between the dsDNA target which contained three CG interruptions and the TFO with three PB units was also confirmed. Additional four-point 2',4'-BNA modifications of the TFO containing three PB units significantly enhanced its triplex-forming ability towards the dsDNA and had a Tm value of 43 degrees C under physiological conditions. These results indicate that a critical inherent problem of TFOs, namely, the sequence limitation of the dsDNA target, may be overcome to a large extent and this should promote antigene applications of TFOs in vitro and in vivo.