Multiphoton near-infrared femtosecond laser pulse-induced DNA damage with and without the photosensitizer proflavine

The excitation of pBR322 supercoiled plasmid DNA with intense near-IR 810 n m fs laser pulses by a simultaneous multiphoton absorption mechanism result s in single-strand breaks after treatment of the irradiated samples with Mi crococcus luteus UV endonuclease. This enzyme cleaves DNA strands at sites of cyclobutane dimers that are formed by the simultaneous absorption of thr ee (or more) 810 nm IR photons (pulse width similar to 140 fs, 76 MHz pulse repetition rate, average power output focused through 10 x microscope obje ctive is similar to 1.2 MW/cm(2)). Direct single-strand breaks (without tre atment with M, luteus) were not observed under these conditions, However, i n the presence of 6 mu M of the intercalator proflavine (PF), both direct s ingle- and double-strand breaks are observed under conditions where substan tial fractions of undamaged supercoiled DNA molecules are still present. Th e fraction of direct double-strand breaks is 30 +/- 5% of all measurable st rand cleavage events, is independent of dosage (up to 6.4 GJ/ cm(2)) and is proportional to I-n, where I is the average power/area of the 810 nm fs la ser pulses, and n = 3 +/- 1, The nicking of two DNA strands in the immediat e vicinity of the excited PF molecules gives rise to this double-strand cle avage. In contrast, excitation of the same samples under low-power, single photon absorption conditions (similar to 400-500 nm) gives rise predominant ly to single-strand breaks, but some double-strand breaks are observed at t he higher dosages, Thus, single-photon excitation with 400-500 nm light and multiphoton activation of PF by near-IR fs laser pulses produces different distributions of single- and double-strand breaks. These results suggest t hat DNA strand cleavage originates from unrelaxed, higher excited states wh en PF is excited by simultaneous IR multiphoton absorption processes.