Steady-state and time-resolved fluorescence studies indicate an unusual conformation of 2-aminopurine within ATAT and TATA duplex DNA sequences.
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 fluorescence 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.
Rai
Priyamvada
P
Biophysics Graduate Group, University of California, Berkeley, CA 94720, USA.
Cole
Timothy David
TD
Thompson
Elizabeth
E
Millar
David P
DP
Linn
Stuart
S
D015075:2-Aminopurine / Q000737:chemistry*; D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013816:Thermodynamics
D009841:Oligonucleotides; D015075:2-Aminopurine; D004247:DNA