Spatially localized generation of nucleotide sequence-specific DNA damage

Psoralens linked to triplex-forming oligonucleotides (psoTFOs) have been us ed in conjunction with laser-induced two-photon excitation (TPE) to damage a specific DNA target sequence. To demonstrate that TPE can initiate photoc hemistry resulting in psoralen-DNA photoadducts, target DNA sequences were incubated with psoTFOs to form triple-helical complexes and then irradiated in liquid solution with pulsed 765-nm laser light, which is half the quant um energy required for conventional one-photon excitation, as used in psora len + UV A radiation (320-400 nm) therapy. Target DNA acquired strand-speci fic psoralen monoadducts in a light dose-dependent fashion. To localize DNA damage in a model tissue-like medium, a DNA-psoTFO mixture was prepared in a polyacrylamide gel and then irradiated with a converging laser beam targ eting the rear of the gel. The highest number of photoadducts formed at the rear while relatively sparing DNA at the front of the gel, demonstrating s patial localization of sequence-specific DNA damage by TPE. To assess wheth er TPE treatment could be extended to cells without significant toxicity, c ultured monolayers of normal human dermal fibroblasts were incubated with t ritium-labeled psoralen without TFO to maximize detectable damage and irrad iated by TPE. DNA from irradiated cells treated with psoralen exhibited a 4 - to 7-fold increase in tritium activity relative to untreated controls. Fu nctional survival assays indicated that the psoralen-TPE treatment was not toxic to cells. These results demonstrate that DNA damage can be simultaneo usly manipulated at the nucleotide level and in three dimensions. This appr oach for targeting photochemical DNA damage may have photochemotherapeutic applications in skin and other optically accessible tissues.