Formation and Quantification of Two-Photon Induced DNA Photolesions

Abstract

The generation of DNA photolesions with a high degree of spatial confinement presents unique opportunities to study the recruitment of UV damage repair proteins to localized damage sites. Photolesion formation is typically accomplished by exposure to UV light which is difficult to manipulate with conventional optics, thus limiting the spatial control over the site of irradiation. As an alternative, we use two-photon absorption of visible light to mimic UV exposure in a form that can be manipulated by conventional optics. We frequency double the output of a tunable Ti:sapphire laser using a barium borate crystal to generate femtosecond pulses of 340-540 nm light. Sample irradiation is performed on 10-20μL volumes confined in a multiwell plate and scanned by a focused beam in a raster pattern through different axial planes. We have adapted a sensitive PCR-based assay to quantify the amount of two-photon induced damage. The assay is premised on a reduction in DNA transcription efficiency by the presence of bulky photolesions; decreased amplification of a sample relative to a control indicates the amount of damage. The assay and laser irradiation system are being tested on linearized pBR322 plasmid, and validated by comparison to direct UV exposure. Our preliminary results indicate that the degree of lesion formation exhibits a nonlinear dependence on power, which is in keeping with the intensity dependence expected for two-photon absorption. Additionally, maximal two-photon DNA damage occurs at wavelength lower than twice the single photon absorption maximum. We are analyzing our results to obtain quantitative information about the yield of photolesions generated by two-photon absorption.