Spatially localized generation of nucleotide sequence-specific DNA damage

Abstract

Psoralens linked to triplex-forming oligonucleotides (psoTFOs) have been used in conjunction with laser-induced two-photon excitation (TPE) to damage a specific DNA target sequence. To demonstrate that TPE can initiate photochemistry 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 quantum energy required for conventional one-photon excitation, as used in psoralen ؉ UV A radiation (320 -400 nm) therapy. Target DNA acquired strand-specific 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 targeting 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 spatial localization of sequence-specific DNA damage by TPE. To assess whether TPE treatment could be extended to cells without significant toxicity, cultured monolayers of normal human dermal fibroblasts were incubated with tritium-labeled psoralen without TFO to maximize detectable damage and irradiated by TPE. DNA from irradiated cells treated with psoralen exhibited a 4-to 7-fold increase in tritium activity relative to untreated controls. Functional survival assays indicated that the psoralen-TPE treatment was not toxic to cells. These results demonstrate that DNA damage can be simultaneously manipulated at the nucleotide level and in three dimensions. This approach for targeting photochemical DNA damage may have photochemotherapeutic applications in skin and other optically accessible tissues.

Combinatorial discovery of two-photon photoremovable protecting groups

“…Two-photon phototherapy with psoralen chromophores has been investigated (3), as has the spatial localization of a psoralen-DNA crosslink (4). Two-photon fluorescence microscopy has provided a new avenue to high spatial resolution in 3D visualization of biological structures (5)(6)(7).…”

Triplex technology in studies of DNA damage, DNA repair, and mutagenesis

“…However, this approach is currently limited because psoralen requires UVA irradiation to form ICLs, and UVA does not penetrate internal organs. Thus, approaches such as the use of laser-induced two-photon excitation, where irradiation at 765 nm is used to photoactivate psoralen, may provide advances for clinical applications [118]. A summary of triplex-mediated induction of recombination in mammalian cells is provided in Table 3.…”

Targeting and processing of site‐specific DNA interstrand crosslinks

“…This may be due in part, to the random mutations induced in the DNA during error-generating repair of the ICLs; however, it is possible to direct psoralen ICLs to specific sites via TFO targeting, which should reduce the off-target effects of PUVA therapy. It has been demonstrated that psoralen-modified TFOs can direct spatially localized ICLs by two-photon excitation using 765 nm light rather than 365 nm (Oh et al 2001). This approach may provide clinical benefit because longer wavelengths of light penetrate deeper into tissue and are less cytotoxic.…”