Real-time observation of DNA repair: 2-aminopurine as a molecular probe

Rajagopal Krishnan; Christina E. Butcher; Dennis H. Oh

doi:10.1117/12.763682

22 February 2008 Real-time observation of DNA repair: 2-aminopurine as a molecular probe

Rajagopal Krishnan, Christina E. Butcher, Dennis H. Oh

Proceedings Volume 6867, Molecular Probes for Biomedical Applications II; 68670M (2008) https://doi.org/10.1117/12.763682
Event: SPIE BiOS, 2008, San Jose, California, United States

Abstract

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.

Citation Download Citation

Rajagopal Krishnan, Christina E. Butcher, and Dennis H. Oh "Real-time observation of DNA repair: 2-aminopurine as a molecular probe", Proc. SPIE 6867, Molecular Probes for Biomedical Applications II, 68670M (22 February 2008); https://doi.org/10.1117/12.763682

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KEYWORDS

Luminescence

Ultraviolet radiation

Therapeutic agents

Quantum efficiency

Skin

Temperature metrology

Tissues

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