Preparation and Characterization of a Deoxyoligonucleotide 49-mer Containing a Site-Specific Thymidylyl-(3’,5')-deoxyadenosine Photoproduct

Irradiation of d(GTATTATG) with 254 nm light gave rise to four major photoproducts, two of which were readily identified by NMR as the cis-syn cyclobutane dimer and the (6-4) photoproduct of the central TT site. Analysis of the NMR data for the other two photoproducts indicated that they were not any of the other known photoproducts of a TT site and might be TA* photoproducts [Bose, S. N., et al. (1983) Science 220, 723-725]. In support of this possibility, the fluorescence spectra of the products of acid hydrolysis of the two photoproducts were very similar to that reported for the hydrolysis product of the TA* photoproduct of TpdA. Only one of the two TA*-containing octamers could be ligated at both ends to form a 49-mer oligonucleotide in the presence of a complementary oligonucleotide scaffold, suggesting that the TA* photoproduct had formed between T5 and A6. The position of the TA* photoproduct was confirmed by mapping the arrest sites for 3'→5’ exonucleolytic degradation of the 49-mer by T4 DNA polymerase and for primer extension opposite the 49-mer by exonuclease deficient Klenow fragment (KF) and Sequenase Version 2.0. The TA* product could also be bypassed by both polymerases, but it was less of a block to KF. Treatment with 1 M aqueous piperidine at 100 °C led to a maximum of about 34% cleavage of the DNA at the site of the TA* product. The ¹H NMR signals of d(GTATTA*TG) were assigned by TOCSY, NOESY, and ¹H-³¹P COSY, and the ¹H NMR shifts for the TA* photoproduct were comparable to those reported for the dinucleotide photoproduct [Koning, T. M. G., et al. (1990) Nucleic Acids Res. 18, 277-284], The biological implications of this photoproduct are also discussed.