Ludovic Le Reste

Conformational transitions in DNA polymerase I revealed by single-molecule FRET

The remarkable fidelity of most DNA polymerases depends on a series of early steps in the reaction pathway which allow the selection of the correct nucleotide substrate, while excluding all incorrect ones, before the enzyme is committed to the chemical step of nucleotide incorporation. The conformational transitions that are involved in these early steps are detectable with a variety of fluorescence assays and include the fingers-closing transition that has been characterized in structural studies. Using DNA polymerase I (Klenow fragment) labeled with both donor and acceptor fluorophores, we have employed single-molecule fluorescence resonance energy transfer to study the polymerase conformational transitions that precede nucleotide addition. Our experiments clearly distinguish the open and closed conformations that predominate in Pol-DNA and Pol-DNA-dNTP complexes, respectively. By contrast, the unliganded polymerase shows a broad distribution of FRET values, indicating a high degree of conformational flexibility in the protein in the absence of its substrates; such flexibility was not anticipated on the basis of the available crystallographic structures. Real-time observation of conformational dynamics showed that most of the unliganded polymerase molecules sample the open and closed conformations in the millisecond timescale. Ternary complexes formed in the presence of mismatched dNTPs or complementary ribonucleotides show unique FRET species, which we suggest are relevant to kinetic checkpoints that discriminate against these incorrect substrates.

Defining the Limits of Single-Molecule FRET Resolution in TIRF Microscopy

Single-molecule FRET (smFRET) has long been used as a molecular ruler for the study of biology on the nanoscale (∼2-10 nm); smFRET in total-internal reflection fluorescence (TIRF) Förster resonance energy transfer (TIRF-FRET) microscopy allows multiple biomolecules to be simultaneously studied with high temporal and spatial resolution. To operate at the limits of resolution of the technique, it is essential to investigate and rigorously quantify the major sources of noise and error; we used theoretical predictions, simulations, advanced image analysis, and detailed characterization of DNA standards to quantify the limits of TIRF-FRET resolution. We present a theoretical description of the major sources of noise, which was in excellent agreement with results for short-timescale smFRET measurements (<200 ms) on individual molecules (as opposed to measurements on an ensemble of single molecules). For longer timescales (>200 ms) on individual molecules, and for FRET distributions obtained from an ensemble of single molecules, we observed significant broadening beyond theoretical predictions; we investigated the causes of this broadening. For measurements on individual molecules, analysis of the experimental noise allows us to predict a maximum resolution of a FRET change of 0.08 with 20-ms temporal resolution, sufficient to directly resolve distance differences equivalent to one DNA basepair separation (0.34 nm). For measurements on ensembles of single molecules, we demonstrate resolution of distance differences of one basepair with 1000-ms temporal resolution, and differences of two basepairs with 80-ms temporal resolution. Our work paves the way for ultra-high-resolution TIRF-FRET studies on many biomolecules, including DNA processing machinery (DNA and RNA polymerases, helicases, etc.), the mechanisms of which are often characterized by distance changes on the scale of one DNA basepair.

Characterization of Dark Quencher Chromophores as Nonfluorescent Acceptors for Single-Molecule FRET

Dark quenchers are chromophores that primarily relax from the excited state to the ground state nonradiatively (i.e., are dark). As a result, they can serve as acceptors for Förster resonance energy transfer experiments without contributing significantly to background in the donor-emission channel, even at high concentrations. Although the advantages of dark quenchers have been exploited for ensemble bioassays, no systematic single-molecule study of dark quenchers has been performed, and little is known about their photophysical properties. Here, we present the first systematic single-molecule study of dark quenchers in conjunction with fluorophores and demonstrate the use of dark quenchers for monitoring multiple interactions and distances in multichromophore systems. Specifically, using double-stranded DNA standards labeled with two fluorophores and a dark quencher (either QSY7 or QSY21), we show that the proximity of a fluorophore and dark quencher can be monitored using the stoichiometry ratio available from alternating laser excitation spectroscopy experiments, either for single molecules diffusing in solution (using a confocal fluorescence) or immobilized on surfaces (using total-internal-reflection fluorescence). The latter experiments allowed characterization of the dark-quencher photophysical properties at the single-molecule level. We also use dark-quenchers to study the affinity and kinetics of binding of DNA Polymerase I (Klenow fragment) to DNA. The measured properties are in excellent agreement with the results of ensemble assays, validating the use of dark quenchers. Because dark-quencher-labeled biomolecules can be used in total-internal-reflection fluorescence experiments at concentrations of 1 μM or more without introducing a significant background, the use of dark quenchers should permit single-molecule Förster resonance energy transfer measurements for the large number of biomolecules that participate in interactions of moderate-to-low affinity.

Red light, green light : probing single molecules using alternating-laser excitation

Single-molecule fluorescence methods, particularly single-molecule FRET (fluorescence resonance energy transfer), have provided novel insights into the structure, interactions and dynamics of biological systems. ALEX (alternating-laser excitation) spectroscopy is a new method that extends single-molecule FRET by providing simultaneous information about structure and stoichiometry; this new information allows the detection of interactions in the absence of FRET and extends the dynamic range of distance measurements that are accessible through FRET. In the present article, we discuss combinations of ALEX with confocal microscopy for studying in-solution and in-gel molecules; we also discuss combining ALEX with TIRF (total internal reflection fluorescence) for studying surface-immobilized molecules. We also highlight applications of ALEX to the study of protein-nucleic acid interactions.

New Technologies for DNA analysis-A review of the READNA Project

The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 41/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3(rd) and 4(th) generation of sequencing methods with nanopores and in situ sequencing, respectively.