A psoralen-conjugated triplex-forming oligodeoxyribonucleotide containing alternating methylphosphonate-phosphodiester linkages: Synthesis and interactions with DNA

A psoralen-conjugated oligodeoxyribopyrimidine (1443), PS-pTTTTCTTTTCTTCTT, where PS is trimethylpsoralen and C is 5-methyl-2'-deoxycytidine, that contains alternating methylphosphonate-phosphodiester internucleotide linkages was synthesized. The ability of 1443 to form triple-stranded complexes with a purine tract in a synthetic DNA duplex was studied. Irradiation of solutions containing the DNA target and 10 mu M 1443 or 0.25 mu M of a similar psoralen-conjugated oligodeoxyribonucleotide that contained all phosphodiester linkages, (1193), with long-wavelength UV light resulted in approximately 80% formation of interstrand cross-links at pH 7.0, 37 degrees C, in the presence of 20 mM magnesium chloride. The extent of tripler formation as monitored by photo-cross-linking decreased over the pH range 5.5-8.0, and the apparent pK of the 5-methylcytosines (C) in 1443 was approximately one-half of a pH unit less than that of the 5-methylcytosines in 1193. Oligomer 1443 formed triplexes in the absence of magnesium, and maximum tripler formation was observed in solutions containing 2.5 mM magnesium, whereas maximal tripler formation by the fully charged 1193 was not observed until the magnesium concentration was 10 mM or higher. Unlike the all-phosphodiester backbone of 1193, the alternating methylphosphonate-phospho diester backbone of 1193 is resistant to hydrolysis by exonucleases in fetal calf serum. The nuclease resistance of 1443 and its ability to form triplexes at very low magnesium concentrations suggests that triplex-forming oligomers with alternating methylphosphonate-phosphodiester backbones may be good candidates for use as antigene reagents in cell culture.