title: Tethered fluorophore motion: studying large DNA conformational changes by single-fluorophore imaging.
abstract: We have previously introduced tethered fluorophore motion (TFM), a single-molecule fluorescence technique that monitors the effective length of a biopolymer such as DNA. TFM uses the same principles as tethered particle motion (TPM) but employs a single fluorophore in place of the bead, allowing TFM to be combined with existing fluorescence techniques on a standard fluorescence microscope. TFM has been previously been used to reveal the mechanism of two site-specific recombinase systems, Cre-loxP and XerCD-dif. In this work, we characterize TFM, focusing on the theoretical basis and potential applications of the technique. Since TFM is limited in observation time and photon count by photobleaching, we present a description of the sources of noise in TFM. Comparing this with Monte Carlo simulations and experimental data, we show that length changes of 100 bp of double-stranded DNA are readily distinguishable using TFM, making it comparable with TPM. We also show that the commonly recommended pixel size for single-molecule fluorescence approximately optimizes signal to noise for TFM experiments, thus enabling facile combination of TFM with other fluorescence techniques, such as Förster resonance energy transfer (FRET). Finally, we apply TFM to determine the polymerization rate of the Klenow fragment of DNA polymerase I, and we demonstrate its combination with FRET to observe synapsis formation by Cre using excitation by a single laser. We hope that TFM will be a useful addition to the single-molecule toolkit, providing excellent insight into protein-nucleic acid interactions.
authors.affiliation: Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Parks road, Oxford, OX1 3PU, UK.
authors.forename: Peter F J
authors.initials: PFJ
authors.lastname: May
authors.affiliation: Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Parks road, Oxford, OX1 3PU, UK.
authors.forename: Justin N M
authors.initials: JNM
authors.lastname: Pinkney
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks road, Oxford, OX1 3QU, UK.
authors.forename: Pawel
authors.initials: P
authors.lastname: Zawadzki
authors.affiliation: Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Parks road, Oxford, OX1 3PU, UK.
authors.forename: Geraint W
authors.initials: GW
authors.lastname: Evans
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks road, Oxford, OX1 3QU, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Sherratt
authors.affiliation: Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Parks road, Oxford, OX1 3PU, UK. Electronic address: a.kapanidis1@physics.ox.ac.uk.
authors.forename: Achillefs N
authors.initials: AN
authors.lastname: Kapanidis
mesh_terms: D003198:Computer Simulation; D004247:DNA / Q000737:chemistry*; D004256:DNA Polymerase I / Q000737:chemistry; D004058:Diffusion; D004926:Escherichia coli; D005456:Fluorescent Dyes / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods*; D008958:Models, Molecular; D009010:Monte Carlo Method; D009038:Motion; D009690:Nucleic Acid Conformation; D058105:Polymerization; D059629:Signal-To-Noise Ratio
chemical_list: D005456:Fluorescent Dyes; D004247:DNA; D004256:DNA Polymerase I