Quantification of Single-Molecule FRET between Quantum Dots and Organic Dyes

Abstract

Single-molecule Förster resonance energy transfer (FRET) is a useful technique for studying inter- and intra-molecular dynamics in biophysics. FRET efficiency is highly sensitive to distance, with half-maximal energy transfer occurring at an inter-dye distance on the order of 5 nm, which makes it possible to quantify distances changes on the molecular scale. FRET efficiency also depends on the spectral overlap and quantum yield of the donor and acceptor molecules, and therefore the choice of dye pair is critical to the success of any experiment. Most organic dyes that are used for single-molecule imaging have bleaching lifetimes in the tens of seconds at most, which limits their use to relatively fast processes. Quantum dot nanocrystals can emit for thousands of seconds without entering a long-lived dark state, which should make them good candidates for use as a FRET donor for longer single-molecule experiments, but there has been little quantitative study of the energy transfer efficiency at the single-molecule level. In this work, we use dsDNA to couple individual quantum dot donors to fluorescent dye acceptors. We then immobilize these FRET pairs on a functionalized glass coverslip and image them using total internal fluorescence (TIRF) microscopy with additional optics that allow us to image the donor and acceptor simultaneously. The length of the DNA between the pair can be varied from 11 to 32 base pairs, allowing us to observe and quantify the energy transfer efficiency over a range of distances on the nanometer scale. Our goal is to develop the use of quantum dots as donors for quantitative single-molecule FRET experiments.