Single-Molecule FRET Detection of Sub-Nanometer Distance Changes in the Range below a 3-Nanometer Scale

Single-molecule fluorescence energy transfer (FRET) detection has become a key technique to monitor intra- and intermolecular distance changes in biological processes. As the sensitive detection range of conventional FRET pairs is limited to 3–8 nm, complement probes are necessary for extending this typical working range. Here, we realized a single-molecule FRET assay for a short distance range of below 3 nm by using a Cy2–Cy7 pair having extremely small spectral overlap. Using two DNA duplexes with a small difference in the labeling position, we demonstrated that our assay can observe subtle changes at a short distance range. High sensitivity in the range of 1–3 nm and compatibility with the conventional FRET assay make this approach useful for understanding dynamics at a short distance.