Direct visualization of enzymatic cleavage and oxidative damage by hydroxyl radicals of single-stranded DNA with a cationic polythiophene derivative

A new method has been developed for the label-free, convenient, and real-time monitoring of the cleavage of single-stranded DNA by single-strand-specific S1 nuclease and hydroxyl radical based on cationic water-soluble poly[ 3-( 3'-N, N, N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrochloride] ( PMNT). The PMNT can form an interpolyelectrolyte complex with ssDNA ( duplex) through electrostatic interactions, in which PMNT takes a highly conjugated and planar conformation, and thus PMNT exhibits a relatively red-shifted absorption wavelength. When ssDNA is hydrolyzed by S1 nuclease or hydroxyl radical into small fragments, the PMNT/ssDNA duplex cannot form. In this case, the PMNT remains in random-coil conformation and exhibits a relatively short absorption wavelength. The nuclease digestion or oxidative damage by hydroxyl radical of DNA can be monitored by absorption spectra or just visualized by the naked-eye in view of the observed PMNT color changes in aqueous solutions. This assay is simple and rapid, and there is no need to label DNA substrates. The most important characteristic of the assay is direct visualization of the DNA cleavage by the naked-eye, which makes it more convenient than other methods that rely on instrumentation. The assay also provides a promising application in drug screening based on the inhibition of oxidative damage of DNA.