The single-stranded deoxynucleotide decamer containing a site-specific Dewar valence isomer of the (6-4) adduct of thymidylyl (3'-->5')-thymidine was generated by direct photolysis of d(CGCATTACGC) with UV-B and UV-C irradiation. The conformation of the Dewar-photomodified deoxyoligonucleotide duplex, (C1-G2-C3-A4-T5[DW]T6-A7-C8-G9-C10) . (G11-C12-G13-T14-A15-A16-T17-G18+ ++-C19-G20), has been studied by one- and two-dimensional NMR spectroscopy. While the eight of the ten complementary nucleotides form Watson-Crick-type hydrogen bonding, the 5'-TpT-3' bases of the Dewar lesion show no evidence of complementary hydrogen bonding formation to each other. The Dewar covalent linkage for the adjacent pyrimidine base leads to unusual base stacking, which is different from that of normal B-DNA. Unusual NOEs indicate that the formation of a Dewar photoproduct in the B-DNA duplex is likely to alter its local and global structures. Also, detailed NMR data show that the base pairing and stacking of the Dewar-photoproduct-containing decamer duplex differ from that of the (6-4)-adduct-containing decamer duplex, suggesting that isomerization of the (6-4) adduct to its Dewar form induces a substantial change in the structure of the oligonucleotide duplex.