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-3 H-diazirin-3-yl)styryl]uridine and its protected phosphoramidite derivatives have been synthesized and introduced into DNA oligomers through solid-phase DNA synthesis. The (trifluoromethyldiazirinyl)stylyl moiety of this nucleoside was found to be sufficiently stable for automated DNA synthesis. In addition, this moiety was found to be stable at 60 degrees C in aqueous solution under the annealing conditions for duplex formation with complementary strands, since >95% of the photolabile nucleoside remained after heating for 1 h. The oligo(dT) 15mer analog bearing the photolabile residue was activated/decomposed by near-UV irradiation. In photoaffinity cross-linking experiments with recombinant rat DNA polymerasebeta, constituted from a 40 kDa polypeptide, using oligo(dT) 15mer analogs bearing the photolabile residue near the 3'-terminus, a covalently bound complex of 45 kDa was obtained in the presence of complementary templates. Thus it was demonstrated that our method for synthesis of photolabile oligodeoxyribonucleotides may be useful for studies of DNA-related enzymes and DNA binding proteins.
Yamaguchi
T
T
Department of Biological Sciences, Teikyo University of Science and Technology, 2525 Yatsuzawa, Uenohara, Yamanashi 409-01, Japan. toyofumi@ntu.ac.jp
Suyama
K
K
Narita
K
K
Kohgo
S
S
Tomikawa
A
A
Saneyoshi
M
M
D000818:Animals; D001389:Azirines / Q000138:chemical synthesis* / Q000737:chemistry; D001483:Base Sequence; D003432:Cross-Linking Reagents; D004256:DNA Polymerase I / Q000378:metabolism*; D004355:Drug Stability; D007202:Indicators and Reagents; D008322:Mammals; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry; D015345:Oligonucleotide Probes / Q000138:chemical synthesis* / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014529:Uridine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry
C106845:2'-deoxy-5-(4-(3-trifluoromethyl-3H-diazirin-3-yl)styryl)uridine; D001389:Azirines; D003432:Cross-Linking Reagents; D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; C027903:oligo (dT); D004256:DNA Polymerase I; D014529:Uridine