Triplex forming oligos (TFOs) that target psoralen photoadducts to specific DNA sequences have generated interest as a
potential agent in gene therapy. TFOs also offer an opportunity to study the mechanism of DNA repair in detail. In an
effort to understand the mechanism of DNA repair at a specific DNA sequence in real-time, we have designed a plasmid
containing a psoralen reaction site adjacent to a TFO binding site corresponding to a sequence within the human
interstitial collagenase gene. Two 2-aminopurine residues incorporated into the purine-rich strand of the TFO binding
site and located within six nucleotides of the psoralen reaction site serve as molecular probes for excision repair events
involving the psoralen photoadducts on that DNA strand. In duplex DNA, the 2-aminopurine fluorescence is quenched.
However, upon thermal or formamide-induced denaturation of duplex DNA to single stranded DNA, the 2-aminopurine
fluorescence increases by eight fold. These results suggest that monitoring 2-aminopurine fluorescence from plasmids
damaged by psoralen TFOs may be a method for measuring excision of single-stranded damaged DNA from the plasmid
in cells. A fluorescence-based molecular probe to the plasmid may significantly simplify the real-time observation of
DNA repair in both populations of cells as well as single cells.