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.

Absorption and fluorescence excitation and emission spectra of the B DNA duplex decamer d[CTGA(2AP)TTCAG]2, where emission from the 2AP (2-aminopurine) base dominates, have been measured as a function of temperature. A low-temperature excitation band in the 260-270-nm region disappears near the duplex melting temperature, Tm = 27 degrees C, but then reappears at higher temperatures. Singlet-singlet energy transfer thus occurs between the normal DNA bases and the 2AP base in the B-helical conformation and to a lesser extent in the structurally-mobile melted conformation. The measured efficiency of transfer is 4-5% at 4 degrees C, near 0 at 30 degrees C, and rises again to 1% at 48 degrees C. Nearest-neighbor-only singlet transfer is likely. Such transfer does not offer a likely explanation for UV damage distributions in DNA.

The sequence, temperature, concentration, and solvent dependence of singlet energy transfer from normal DNA bases to the 2-aminopurine base in synthesized DNA oligomers were investigated by optical spectroscopy. Transfer was shown directly by a variable fluorescence excitation band at 260-280 nm. Adenine (A) is the most efficient energy donor by an order of magnitude. Stacks of A adjacent to 2AP act as an antenna for 2AP excitation. An interposed G, C, or T base between A and 2AP effectively blocks transfer from A to 2AP. Base stacking facilitates transfer, while base pairing reduces energy transfer slightly. The efficiency is differentially temperature dependent in single- and double-stranded oligomers and is highest below 0 degrees C in samples measured. An efficiency transition occurs well below the melting transition of a double-stranded decamer. The transfer efficiency in the duplex decamer d(CTGA[2AP]TTCAG)(2) is moderately dependent on the sample and salt concentration and is solvent dependent. Transfer at physiological temperature over more than a few bases is improbable, except along consecutive A's, indicating that singlet energy transfer is not a major factor in the localization of UV damage in DNA. These results have features in common with recently observed electron transfer from 2AP to G in oligonucleotides.

2-Aminopurine (2AP) absorption and fluorescence excitation and emission spectra in a series of solvents have been measured to assess effects of solvent polarity. Emission spectra of the free base shift to the red in solvents of a higher dielectric constant, including water but excepting dioxane. Excitation spectra also red-shift, except in water. A change in dipole moment of 2.8 D upon excitation is obtained from a Bilot-Kawski plot which includes data from potentially anomolous solvents such as alcohols but which excludes dioxane and aqueous solvents. Attachment of ribose or 2'-deoxyribose causes 1 to 2-nm shifts in the position of fluorescence excitation and emission spectra of 2AP in water and little change in fluorescence yield. Melting of the DNA duplex d[CTGA(2AP)TTCAG]2 yields a blue shift of the excitation and no shift of the emission spectrum-results consistent with increased exposure to water and formation of 2AP-water H bonds in the ground state. The spectral shift occurs 5°C or more below the helix melting temperature, implying a premelting structural change in the decamer.

Time-resolved fluorescence decay of a single-stranded DNA decamer d(CTGAAT5CAG), where d5 is the fluorescent base 1-(beta-D-2'-deoxyribosyl)-5-methyl-2-pyrimidinone, was measured and analyzed at several temperatures. The d5 base in the decamer is resolved into three states according to their fluorescence decay lifetime characteristics and temperature dependence of their associated amplitudes: fully extended and completely unstacked state, loosely associated state, and fully stacked state. These states are in slow exchange compared to their fluorescence decay rates. The population of the fully extended and completely unstacked state is small and decreases further with increasing temperature. The loosely associated state, whose fluorescence can still be efficiently quenched by other DNA bases, occupies a large portion of the conventionally defined unstacked state. Stacking enthalpy and entropy for the d5 base with thymine or cytosine bases in the DNA decamer are calculated to be -6.6 kcal/mol and -22 cal/mol.K, respectively. This work shows that fluorescent bases in DNA can be useful to the study of local conformations of bases.