Nucleic Acids Research, 2003, Vol. 31, No. 9 2323-2332
© 2003 Oxford University Press
Steady-state and time-resolved fluorescence studies indicate an unusual conformation of 2-aminopurine within ATAT and TATA duplex DNA sequences
Biophysics Graduate Group, University of California, Berkeley, CA 94720, USA, 1 Department of Molecular Biology, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA and 2 Division of Biochemistry and Molecular Biology, Barker Hall, University of California, Berkeley, CA 94720-3202, USA
Timothy David Cole, Telik Inc., 750 Gateway Boulevard, San Francisco, CA 94080, USA
2-Aminopurine (2-AP), a fluorescent analog of adenine, has been widely used as a probe for local DNA conformation, since excitation and emission characteristics and fluoresence lifetimes of 2-AP vary in a sequence-dependent manner within DNA. Using steady-state and time-resolved fluorescence techniques, we report that 2-AP appears to be unusually stacked in the internal positions of ATAT and TATA in duplex DNA. The excitation wavelength maxima for 2-AP within these contexts were red shifted, indicating reduced solvent exposure for the fluorophore. Furthermore, in these contexts, 2-AP fluorescence was resistant to acrylamide-dependent collisional quenching, suggesting that the fluorophore is protected by its stacked position within the duplex. This conclusion was further reinforced by the presence of a secondary peak at 275 nm in the fluorescence excitation spectra that is indicative of efficient excitation energy transfer from nearby non-fluorescent DNA bases. Fluorescence anisotropy decay and internal angular ‘wobbling’ motion measurements of 2-AP within these alternating AT contexts were also consistent with the fluorophore being highly constrained and immobile within the base stack. When these fluorescence characteristics are compared with those of 2-AP within other duplex DNA sequence contexts, they are unique.
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