We designed a novel 2′- O,5′- N bridged nucleic acid, 2′,5′-BNA ON, whose sugar puckering was fixed to S-type conformation by an N– O linkage. A dimer unit formed from 2′,5′-BNA ON-U and thymidine was synthesized via a coupling reaction between a protected 2′,5′-BNA ON-U monomer and a thymidine derivative. Introduction of 2′,5′-BNA ON-U into DNA was carried out using conventional phosphoramidite chemistry with a DNA synthesizer. The hybridization abilities of 2′,5′-BNA ON-U-modified oligonucleotides against DNA or RNA complement were evaluated.