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 complementary sequences compared to those with single base mismatches, consistent with selective photoinduced dehybridization of partially mismatched sequences. The demonstration of photoinduced differential dehybridization phenomenon on chip surfaces in the presence of fluorophores indicates that photonic DNA hybridization stringency is compatible with optical readouts in heterogeneous assays.