SYNTHESIS AND EVALUATION OF OLIGODEOXYRIBONUCLEOTIDES CONTAINING AN ARYL(TRIFLUOROMETHYL)DIAZIRINE MOIETY AS THE CROSS-LINKING PROBE - PHOTOAFFINITY-LABELING OF MAMMALIAN DNA-POLYMERASE-BETA

Photolabile 2'-deoxy-E-5-[4-(3-trifluoromethyl-3H-diazirin -3-yl)styry l]uridine and its protected phosphoramidite derivatives have been synt hesized and introduced into DNA oligomers through solid-phase DNA synt hesis. The (trifluoromethyldiazirinyl)stylyl moiety of this nucleoside was found to be sufficiently stable for automated DNA synthesis. In a ddition, this moiety was found to be stable at 60 degrees C in aqueous solution under the annealing conditions for duplex formation with com plementary strands, since >95% of the photolabile nucleoside remained after heating for 1 h. The oligo(dT) 15mer analog bearing the photolab ile residue was activated/decomposed by near-UV irradiation. In photoa ffinity cross-linking experiments with recombinant rat DNA polymerase beta, constituted from a 40 kDa polypeptide, using oligo(dT) 15mer ana logs bearing the photolabile residue near the 3'-terminus, a covalentl y bound complex of 45 kDa was obtained in the presence of complementar y templates. Thus it was demonstrated that our method for synthesis of photolabile oligodeoxyribonucleotides may be useful for studies of DN A-related enzymes and DNA binding proteins.