Characterization of individual adenine residues in DNA by a combination ofsite-selective C8-deuteration and UV resonance Raman difference spectroscopy

Raman spectroscopy has been combined with site-selective isotopic labeling techniques to obtain structural information on a selected nucleic acid resi due in oligonucleotides. In the difference spectrum between the unlabeled a nd site-selectively labeled oligonucleotides, the Raman signals from the re sidue at the labeled position show up, while the signals from the residues at unlabeled positions are canceled out. To demonstrate the utility of this new method, we have prepared a self-complementary tetradecamer DNA, d(AGTG CTCGAGCACT)(2), containing single C8-deuterated adenine at position 9 (A9) or 12 (A12). UV (257 nm) resonance Raman difference spectra between the unl abeled and labeled oligonucleotides in solution reveal slightly different m icroenvironments around A9 and A 12 as expected for a double-stranded helic al structure of the tetradecamer DNA. In the presence of an antitumor antib iotic, actinomycin D, which intercalates into the 5'-GC-3' sequence of DNA, the Raman signals of A9 on the 5'-side of the intercalation site become si gnificantly weaker, indicating an increased base stacking with adjacent gua nine bases. In contrast, the Raman signals of A12 on the 3'-side are not af fected by the binding of actinomycin D. This observation provides the first experimental evidence that the intercalation of actinomycin D induces an a symmetric structural alteration of DNA in solution. (C) 2001 Elsevier Scien ce B.V. All rights reserved.