Melting and premelting transitions of an oligomer measured by DNA base fluorescence and absorption.
Incorporation of 2-aminopurine (2AP) in place of adenine gives an optical probe of local and global DNA conformation. The temperature dependence of the absorption of the duplex d[CTGA(2AP)-TTCAG]2 DNA decamer shows that the helix has approximately an all-or-none melting transition. Absorbance at wavelengths of 260 and 330 nm monitors the average normal base conformation and the 2AP base local conformation, respectively. From this measure, the 2AP base melts less than 1 degree C below the other bases. Temperature-dependent lifetime measurements of 2AP also mirror the melting transition. Absorption spectra show that below Tm most 2AP's are H-bonded. Fluorescence intensity and excitation spectra measurements show, on the other hand, that the most highly-fluorescent states correspond to non-H-bonded 2AP's which sense conformational changes of the helix. The temperature dependence of the fluorescence spectral shift shows the conformation and/or dynamics of the 2AP base changes 10 degrees C or more below Tm. The data suggest a premelting transition which is purely dynamic in nature--transient exposure of most 2AP's to water increases, while the average conformation remains B-helical.
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Department of Physics, University of Alabama at Birmingham 35294-1170.
Evans
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Nordlund
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D015075:2-Aminopurine / Q000737:chemistry; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D015335:Molecular Probes; D009838:Oligodeoxyribonucleotides; D015075:2-Aminopurine; D004247:DNA