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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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.

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In an effort to increase our understanding of the molecular rearrangements that occur during lipid bilayer fusion, we have used different fluorescent probes to characterize the lipid rearrangements associated with poly(ethylene glycol) (PEG)-mediated fusion of DOPC:DL(18:3)PC (85:15) small, unilamellar vesicles (SUVs). Unlike in our previous studies of fusion kinetics [Lee, J., and Lentz, B. R., Biochemistry 36, 6251-6259], these vesicles have mean diameters of 20 nm compared to 45 nm. Surprisingly, we found significant inter-vesicle lipid mixing at 5 wt % PEG, well below the PEG concentration required (17.5 wt %) for vesicles fusion. Lipid movement rate between bilayers (or inter-leaflet movement) increased abruptly at 10 wt % PEG, and the rate of lipid mixing increased thereafter with increasing amounts of PEG. The characteristic time of lipid mixing between outer leaflets (tau approximately equal to 24 s) was comparable to that observed at and above PEG concentrations needed to induce fusion (17.5 wt %) of either 20 or 45 nm vesicles. We also found that slower lipid mixing (tau approximately equal to 267 s) between fusing vesicles occurred on the same time scale or slightly faster than vesicle contents mixing (tau approximately equal to 351 s). In addition, our measurements showed that lipids redistributed across the bilayer on a time scale just slightly faster than pore formation (tau approximately equal to 217 s). This is the first demonstration of trans-bilayer movement of lipids during fusion. We also found that water was excluded from the bilayer (tau approximately equal to 475 s) during product maturation. These observations suggest that fusion in smaller vesicles (approximately 20 nm) proceeds via a multistep mechanism similar to that we reported for somewhat larger vesicles, except that two intermediates are no longer clearly resolved.