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Three-Dimensional
Targeting of Photodynamic Damage to Specific DNA Sequences
Oh, Dennis1,2,
King, Brett3, Boxer, Steven3
and Hanawalt, Philip4
Department of Dermatology, University of California, San Francisco1
San Francisco VA Medical Center2
Department of Chemistry, Stanford University3
Department of Biological Sciences, Stanford University4
Abstract-
DNA damaging agents used in dermatology do not typically discriminate
among the many targets found in the structurally and functionally distinct
architecture of skin. We have previously used triple helix-forming oligonucleotides
linked to psoralen (psoTFOs) to damage specific nucleotide sequences
of the human collagenase gene. This method has now been combined with
laser-induced two-photon excitation (TPE) to damage a specific sequence
of DNA residing in a specific location in three-dimensional space. First,
to demonstrate TPE-induced photoadducts, collagenase DNA target sequences
were incubated with psoTFOs and then irradiated in liquid solution with
pulsed 765 nm laser light which is half of the quantum energy needed
for conventional one photon excitation used in psoralen + UVA (PUVA)
therapy. The DNA target acquired strand-specific psoralen monoadducts
in a light dose-dependent fashion. Second, to simulate DNA damage in
a tissue, the DNA-psoTFO mixture was formed in a polyacrylamide gel
that was 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 preferential
localization of sequence-specific DNA damage in 3D space. Third, to
assess whether TPE treatment could be extended to cells, monolayers
of human dermal fibroblasts were incubated with tritium-labeled psoralen,
without TFO in order to maximize detectable damage, and irradiated with
laser TPE. DNA from cells treated with psoralen and irradiated had a
seven-fold increase in tritium-activity relative to untreated controls.
Survival assays indicated the psoralen-TPE treatment was not acutely
toxic. These results demonstrate that DNA damage can be simultaneously
manipulated at the nucleotide level and in three-dimensional space.
This approach should be useful for dissecting whole tissue responses
to DNA damage and for gene-specific photodynamic therapy.
Keywords: psoralen,
laser, photodynamic therapy, DNA damage
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