Phototriggered bod cleavage has found wide application in chemistry as well as in biology. Nevertheless, there are only a few methods available for site-specific photochemical induction of DNA strand scission despite numerous potential applications. In this study we report the development of new photocleavable nucleotides based on the photochemistry of o-nitrobenzyl esters. The light-sensitive moieties were generated through introduction of o-nitrophenyl groups at the 5'C position of the nucleoside sugar backbone. The newly synthesized, modified nucleosides were incorporated in oligonucleotides and are able to build stable DNA duplexes. In such a way modified oligonucleotides ca cleaved site-specifically upon irradiation with > 360 nm light with high efficiency. Furthermore, we show that these modifications can be bypassed in DNA synthesis promoted by Thermus aquaticus DNA polymerase.