Photocontrolled DNA Hybridization Stringency with Fluorescence Detection in Heterogeneous Assays

Abstract

We study hybridization and light-induced dehybridization of azobenzene-modified DNA bound to glass substrates with fluorescently labeled oligonucleotide targets in solution. We show that fluorescent readout using a commercial array scanner is compatible with azobenzene-modified DNA capture sequences and, importantly, that the fluorescent signals generated using azobenzene-modified sequences are similar to those from azobenzene-free capture strands. In addition, we demonstrate that we can photoswitch azobenzene molecules on a surface in the presence of fluorophores and thus that we are able to control the dehybridization behavior of the immobilized azobenzene-modified DNA with its target sequence in solution. We further study the dehybridization of perfectly matched target sequences and the single-base-mismatched sequences as a function of radiant fluence. Though both target sequences dehybridize upon exposure to ultraviolet light, we measure higher fluorescent signals after UV irradiation for perfectly co...