Professor Achillefs Kapanidis

Abstract:

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.

Abstract:

Many biological processes, such as gene transcription and replication, involve opening and closing of short regions of double-stranded DNA (dsDNA). Few techniques, however, can study these processes in real time or at the single-molecule level. Here, we present a Förster resonance energy transfer (FRET) assay that monitors the state of DNA (double- vs single-stranded) at a specific region within a DNA fragment, at both the ensemble level and the single-molecule level. The assay utilizes two closely spaced fluorophores: a FRET donor fluorophore (Cy3B) on the first DNA strand and a FRET acceptor fluorophore (ATTO647N) on the complementary strand. Because our assay is based on quenching and dequenching FRET processes, i.e., the presence or absence of contact-induced fluorescence quenching, we have named it a "quenchable FRET" assay or "quFRET". Using lac promoter DNA fragments, quFRET allowed us to sense transcription bubble expansion and compaction during abortive initiation by bacterial RNA polymerase. We also used quFRET to confirm the mode of action of gp2 (a phage-encoded protein that acts as a potent inhibitor of Escherichia coli transcription) and rifampicin (an antibiotic that blocks transcription initiation). Our results demonstrate that quFRET should find numerous applications in many processes involving DNA opening and closing, as well as in the development of new antibacterial therapies involving transcription.

Abstract:

The analysis of structure and dynamics of biomolecules is important for understanding their function. Toward this aim, we introduce a method called 'switchable FRET', which combines single-molecule fluorescence resonance energy transfer (FRET) with reversible photoswitching of fluorophores. Typically, single-molecule FRET is measured within a single donor-acceptor pair and reports on only one distance. Although multipair FRET approaches that monitor multiple distances have been developed, they are technically challenging and difficult to extend, mainly because of their reliance on spectrally distinct acceptors. In contrast, switchable FRET sequentially probes FRET between a single donor and spectrally identical photoswitchable acceptors, dramatically reducing the experimental and analytical complexity and enabling direct monitoring of multiple distances. Our experiments on DNA molecules, a protein-DNA complex and dynamic Holliday junctions demonstrate the potential of switchable FRET for studying dynamic, multicomponent biomolecules.

Abstract:

Single-molecule fluorescence microscopy is currently one of the most popular methods in the single-molecule toolbox. In this review, we discuss recent advances in fluorescence instrumentation and assays: these methods are characterized by a substantial increase in complexity of the instrumentation or biological samples involved. Specifically, we describe new multi-laser and multi-colour fluorescence spectroscopy and imaging techniques, super-resolution microscopy imaging and the development of instruments that combine fluorescence detection with other single-molecule methods such as force spectroscopy. We also highlight two pivotal developments in basic and applied biosciences: the new information available from detection of single molecules in single biological cells and exciting developments in fluorescence-based single-molecule DNA sequencing.

Abstract:

Probability distribution analysis (PDA) is a recently developed statistical tool for predicting the shapes of single-molecule fluorescence resonance energy transfer (smFRET) histograms, which allows the identification of single or multiple static molecular species within a single histogram. We used a generalized PDA method to predict the shapes of FRET histograms for molecules interconverting dynamically between multiple states. This method is tested on a series of model systems, including both static DNA fragments and dynamic DNA hairpins. By fitting the shape of this expected distribution to experimental data, the timescale of hairpin conformational fluctuations can be recovered, in good agreement with earlier published results obtained using different techniques. This method is also applied to studying the conformational fluctuations in the unliganded Klenow fragment (KF) of Escherichia coli DNA polymerase I, which allows both confirmation of the consistency of a simple, two-state kinetic model with the observed smFRET distribution of unliganded KF and extraction of a millisecond fluctuation timescale, in good agreement with rates reported elsewhere. We expect this method to be useful in extracting rates from processes exhibiting dynamic FRET, and in hypothesis-testing models of conformational dynamics against experimental data.