Investigation of DNA conformation and sequence by controlling the fluorescence blinking

Abstract

In order to achieve ultralow detection limit, one strategy would be to focus on a detection method that relies on the properties of molecules that become highlighted when we look at molecules at the single-molecule level. On the single-molecule level, fluorescent molecules often exhibit fluctuating emissions between bright ‘‘on’’ and dark “off” states, so-called “blinking.” During the repetitive cycles of excitation and emission, fluorescent molecules may occasionally enter non-fluorescent off states, such as a triplet state, a radical ion state, and an isomerized state. Reversible formation of such off states causes a blinking of the fluorescence. The changes in the surrounding local microenvironment that modulate the duration of these off state (= τ_OFF) would be detectable at the single-molecule level by monitoring of the fluorescence blinking. In this study, we demonstrate that DNA conformations such as a hairpin, a duplex, and a triplex, and single nucleotide changes in DNA sequences can be monitored by controlling the fluorescence blinking.