Fluorescence Redox Blinking Adaptable to Structural Analysis of Nucleic Acids

The phenomenon of blinking is unique to single‐molecule fluorescence measurements. By designing a fluorophore with an appropriate dark‐state lifetime τoff, a kinetic analysis based on the control of fluorescence blinking (KACB) was devised to investigate the dynamics of biomolecules. By controlling the redox‐reaction‐based blinking (rKACB), conformational dynamics of RNA at the single‐molecule level was previously investigated. However, there is little knowledge about suitable fluorescent molecules for rKACB, and the application of rKACB has been limited to the analysis of hairpins and duplex structures of nucleic acids. In this work, various fluorescent molecules, including Alexa 488, R6G, TAMRA, ATTO 647N and ATTO 655, were evaluated for rKACB. Moreover, rKACB was adapted to the discrimination of DNA/DNA and DNA/RNA nucleic acid duplexes and investigation of antigen–antibody interactions. By changing the size of the oxidant, it was possible to determine the solvent accessibility of the target domain of the analyzed biomolecules. Analysis in a blink: Kinetic analysis based on the control of fluorescence blinking utilizing redox reactions (rKACB) was investigated for various fluorescent molecules and for different oxidant sizes (see figure). rKACB was shown to be adaptable to the discrimination of B‐form DNA/DNA and A‐like DNA/RNA helixes, as well as to single‐molecule measurements of antigen–antibody interactions.