2-WAVELENGTH FEMTOSECOND LASER-INDUCED DNA-PROTEIN CROSS-LINKING

Nucleic acid-protein interactions are essential for storage, reproduct ion and expression of genetic information. Biochemical methods, such a s dimethyl sulfate genomic footprinting, have been developed to study stable protein-DNA interactions in vivo and chemical crosslinking has been used for less stable interactions, but the chemical agents are sl ow, damage cells and perturb native equilibria, To avoid these perturb ations, UV laser crosslinking offers an alternative, although the ener gies required for significant crosslinking cause extensive DNA damage. We find that a combination of femtosecond laser pulses at two differe nt wavelengths, in the UV and the visible range, increases the crossli nking efficiency while minimizing DNA damage. This technique also allo wed us to directly measure the singlet S-1 lifetime of native DNA (tau (S1) = 3.2 +/- 0.2 ps), which is mainly determined by the lifetime of thymine [tau(S1) = 2.8 +/- 0.4 ps for (dT)(16)], the photochemically m ost reactive base. Our results suggest that two wavelength femtosecond laser pulses are well suited for the identification of transcription factors interacting with defined sequences and for studying the kineti cs of protein-nucleic acid interactions in intact cells.