Designing Distance Standards for Biomolecular Single-Molecule FRET

Single-molecule FÓ§rster Resonance Energy Transfer (smFRET) has become a powerful tool for investigating the structural, energetic, and dynamic aspects of biological macromolecules. FRET is considered a nanoscale spectroscopic ruler in which the distance between donor and acceptor fluorophores coupled to a biomolecule of interest can be determined with high precision based on the efficiency of energy transfer between them. However, several factors can influence the efficiency of energy transfer and thus the overall accuracy of the distance measurement. This includes poor alignment of optical components in the microscopy system and improper correction factors associated with the excitation lasers, fluorophores, and detectors. To address this issue, we engineered several simple fluorescently labeled DNA constructs that can be easily utilized to determine both the alignment of optical components as well as the necessary instrumental correction factors. These constructs are comprised of three DNA oligonucleotide sequences: two probe oligonucleotides labeled with donor, Cy3B, and acceptor, CF660R, fluorophores that specifically bind to the complementary ends of the target oligonucleotide. Five different lengths of the target oligonucleotide were chosen to obtain five constructs with varying donor-acceptor distances. Then we used smFRET to measure the inter-fluorophore distances of these constructs. First, we confirmed that these constructs give rise to different inter-fluorophore distances as observed by distinct average transfer efficiencies. Second, we tested our ability to resolve the physical mixtures of different constructs. Due to the simplicity of these constructs, measurements can be performed on a routine basis to evaluate the performance and stability of the microscope. Finally, the modular design of our fluorescently labeled DNA constructs can be used in the future to evaluate the performance of different donor and acceptor fluorophores.