Molecular beacon probes of oligonucleotides photodamaged by Psoralen

Abstract

Ultraviolet A (UVA)-irradiated 4′-hydroxymethyl-4,5′,8- trimethyl psoralen (HMT) in the presence of a poly-dT 17 and dA 7TTA 8 oligonucleotides produces HMT-dT 17 and HMT-dA 7TTA 8 adducts in aqueous solution. In this article, we determine whether these HMT-dT 17 and HMT-dA 7TTA 8 adducts can be detected with a molecular beacon (MB) probe. We measure the degree of damage in dT 17 and dA 7TTA 8 solutions containing UVA-activated HMT via monitoring the decrease in MB fluorescence. Photoproduct formation is confirmed by MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-fight mass spectrometry measurements) and absorption spectroscopy. The MB fluorescence decreases upon UVA irradiation in the presence of HMT with a single-exponential time constants of 114.2 ± 6.5 min for HMT-dT 17 adducts and 677.8 ± 181.8 min for HMT-dA 7TTA 8 adducts. Our results show that fluorescent MB probes are a selective, robust and accurate tool for detecting UVA-activated HMT-induced DNA damage. Early, easy chemically induced DNA damage detection may help prevent disease and aid cancer treatment. Molecular beacon probes can be used for the detection of chemically induced DNA damage due to their sensitivity and specificity. This article highlights the ability of molecular beacons to detect DNA damage induced by psoralen, a phototherapeutic drug that upon exposure to UVA light forms adducts with both ssDNA and dsDNA. The molecular beacons provide a rapid, mix-and-read assay that can discriminate between undamaged and damaged ssDNA without the need to isolate the damaged ssDNA from the solution nor from the damage agent. © 2012 The American Society of Photobiology.