Probing the structure and dynamics of a DNA hairpin by ultrafast quenching and fluorescence depolarization.

DNA hairpins have been investigated in which individual adenines were replaced by their fluorescent analog 2-aminopurine (2AP). The temperature dependence of the time evolution of polarized emission spectra was monitored with picosecond time resolution. Four isotropic decay components for each oligonucleotide indicated the coexistence of at least four conformations. The fluorescence for three of these was significantly quenched, which is explained by hole transfer from 2AP to guanine(s). An approximately 8-ps component is ascribed to direct hole transfer, the approximately 50-ps and approximately 500-ps components are ascribed to structural reorganization, preceding hole transfer. At room temperature, a fraction remains unquenched on a 10-ns timescale, in contrast to higher temperatures, where the flexibility increases. Besides quenching due to base stacking, a second quenching process was needed to describe the data. Evidence for both intrastrand and interstrand hole transfer was found. The extracted probability for stacking between neighboring bases in double-stranded regions was estimated to be approximately 75% at room temperature and approximately 25% at 80 degrees C, demonstrating structural disorder of the DNA. Fluorescence depolarization revealed both local dynamics of the DNA and overall dynamics of the entire oligonucleotide. Upon raising the temperature, the C-N terminus of the hairpin appears to melt first; the rest of the hairpin denatures above the average melting temperature.