Single-molecule analysis of DNA replication in Xenopus egg extracts

The recent advent in single-molecule imaging and manipulation methods has made a significant impact on the understanding of molecular mechanisms underlying many essential cellular processes. Single-molecule techniques such as electron microscopy and DNA fiber assays have been employed to study the duplication of genome in eukaryotes. Here, we describe a single-molecule assay that allows replication of DNA attached to the functionalized surface of a microfluidic flow cell in a soluble Xenopus leavis egg extract replication system and subsequent visualization of replication products via fluorescence microscopy. We also explain a method for detection of replication proteins, through fluorescently labeled antibodies, on partially replicated DNA immobilized at both ends to the surface.     

High-throughput single-molecule studies of protein–DNA interactions

Fluorescence and force-based single-molecule studies of protein–nucleic acid interactions continue to shed critical insights into many aspects of DNA and RNA processing. As single-molecule assays are inherently low-throughput, obtaining statistically relevant datasets remains a major challenge. Additionally, most fluorescence-based single-molecule particle-tracking assays are limited to observing fluorescent proteins that are in the low-nanomolar range, as spurious background signals predominate at higher fluorophore concentrations. These technical limitations have traditionally limited the types of questions that could be addressed via single-molecule methods. In this review, we describe new approaches for high-throughput and high-concentration single-molecule biochemical studies. We conclude with a discussion of outstanding challenges for the single-molecule biologist and how these challenges can be tackled to further approach the biochemical complexity of the cell.     

Single-molecule perspectives on helicase mechanisms and functions

Helicases are a diverse group of molecular motors that utilize energy derived from the hydrolysis of nucleoside triphosphates (NTPs) to unwind and translocate along nucleic acids. These enzymes play critical roles in nearly all aspects of nucleic acid metabolism, and consequently, a detailed understanding of helicase mechanisms at the molecular level is essential. Over the past few decades, single-molecule techniques, such as optical tweezers, magnetic tweezers, laminar flow, fluorescence resonance energy transfer (FRET), and DNA curtains, have proved to be powerful tools to investigate the functional properties of both DNA and RNA helicases. These approaches allow researchers to manipulate single helicase molecules, perturb their free energy landscape to probe the chemo-mechanical activities of these motors, and to detect the conformational changes of helicases during unwinding. Furthermore, these techniques also provide the capability to distinguish helicase heterogeneity and monitor helicase motion at nanometer spatial and millisecond temporal resolutions, ultimately providing new insights into the mechanisms that could not be resolved by ensemble assays. This review outlines the single-molecule techniques that have been utilized for measurements of helicase activities and discusses helicase mechanisms with a focus on functional and mechanistic insights revealed through single-molecule investigations in the past five years.     

Super-resolution Microscopy Approaches for Live Cell Imaging

By delivering optical images with spatial resolutions below the diffraction limit, several super-resolution fluorescence microscopy techniques opened new opportunities to study biological structures with details approaching molecular structure sizes. They have now become methods of choice for imaging proteins and their nanoscale dynamic organizations in live cells. In this mini-review, we describe and compare the main far-field super-resolution approaches that allow studying endogenous or overexpressed proteins in live cells.     

Visualizing the Behavior of Human Rad51 at the Single-Molecule Level

The repair of double-stranded DNA breaks by homologous recombination is essential for maintaining genome integrity. Much of what we know about this DNA repair pathway in eukaryotes has been gleaned from genetics, in vivo experiments with GFP-tagged proteins and traditional biochemical experiments with purified proteins. However, many questions have remained inaccessible to these experimental approaches. Recent technological advances have made it possible to directly visualize the behaviors of individual DNA and protein molecules in vitro, and it is now becoming feasible to apply these technology-driven approaches to complex biochemical systems, such as those involved in the repair of damaged DNA. This report summarizes the use of total internal reflection fluorescence microscopy to probe fundamental aspects of protein-DNA interactions at the single-molecule level, and specific emphasis is placed on our efforts to develop new methods and techniques for studying DNA repair. Using these new approaches we are investigating the DNA-binding behavior of human Rad51 and we have recently demonstrated that this protein can slide on dsDNA via a one-dimensional random walk mechanism driven solely by thermal fluctuations of the surrounding solvent. Here, we highlight some possible implications of this recent finding, and we also briefly discuss the potential benefits of future single-molecule studies in the study of protein-DNA interactions and DNA repair.     

Single-molecule fluorescence imaging: Generating insights into molecular interactions in virology

Single-molecule fluorescence methods remain a challenging yet information-rich set of techniques that allow one to probe the dynamics, stoichiometry and conformation of biomolecules one molecule at a time. Viruses are small (nanometers) in size, can achieve cellular infections with a small number of virions and their lifecycle is inherently heterogeneous with a large number of structural and functional intermediates. Single-molecule measurements that reveal the complete distribution of properties rather than the average can hence reveal new insights into virus infections and biology that are inaccessible otherwise. This article highlights some of the methods and recent applications of single-molecule fluorescence in the field of virology. Here, we have focused on new findings in virus–cell interaction, virus cell entry and transport, viral membrane fusion, genome release, replication, translation, assembly, genome packaging, egress and interaction with host immune proteins that underline the advantage of single-molecule approach to the question at hand. Finally, we discuss the challenges, outlook and potential areas for improvement and future use of single-molecule fluorescence that could further aid our understanding of viruses.     

Super-resolution Microscopy Approaches for Live Cell Imaging

By delivering optical images with spatial resolutions below the diffraction limit, several super-resolution fluorescence microscopy techniques opened new opportunities to study biological structures with details approaching molecular structure sizes. They have now become methods of choice for imaging proteins and their nanoscale dynamic organizations in live cells. In this mini-review, we describe and compare the main far-field super-resolution approaches that allow studying endogenous or overexpressed proteins in live cells.     

Site-specific labeling of Saccharomyces cerevisiae ribosomes for single-molecule manipulations

Site-specific labeling of Escherichia coli ribosomes has allowed application of single-molecule fluorescence spectroscopy and force methods to probe the mechanism of translation. To apply these approaches to eukaryotic translation, eukaryotic ribosomes must be specifically labeled with fluorescent labels and molecular handles. Here, we describe preparation and labeling of the small and large yeast ribosomal subunits. Phylogenetically variable hairpin loops in ribosomal RNA are mutated to allow hybridization of oligonucleotides to mutant ribosomes. We demonstrate specific labeling of the ribosomal subunits, and their use in single-molecule fluorescence and force experiments.     

Single-molecule approaches to characterizing kinetics of biomolecular interactions

Single-molecule fluorescence techniques have emerged as powerful tools to study biological processes at the molecular level. This review describes the application of these methods to the characterization of the kinetics of interaction between biomolecules. A large number of single-molecule assays have been developed that visualize association and dissociation kinetics in vitro by fluorescently labeling binding partners and observing their interactions over time. Even though recent progress has been significant, there are certain limitations to this approach. To allow the observation of individual, fluorescently labeled molecules requires low, nanomolar concentrations. I will discuss how such concentration requirements in single-molecule experiments limit their applicability to investigate intermolecular interactions and how recent technical advances deal with this issue.     

Different approaches for assaying melanosome transfer

Many approaches have been tried to establish assays for melanosome transfer to keratinocytes. In this report, we describe and summarize various novel attempts to label melanosomes in search of a reliable, specific, reproducible and quantitative assay system. We tried to fluorescently label melanosomes by transfection of GFP-labeled melanosomal proteins and by incubation of melanocytes with fluorescent melanin intermediates or homologues. In most cases a weak cytoplasmic fluorescence was perceived, which was probably because of incorrect sorting or deficient incorporation of the fluorescent protein and different localization. We were able to label melanosomes via incorporation of 14C-thiouracil into melanin. Consequently, we tried to develop an assay to separate keratinocytes with transferred radioactivity from melanocytes after co-culture. Differential trypsinization and different magnetic bead separation techniques were tested with unsatisfactory results. An attempt was also made to incorporate fluorescent thiouracil, since this would allow cells to be separated by FACS. In conclusion, different methods to measure pigment transfer between donor melanocytes and acceptor keratinocytes were thoroughly examined. This information could give other researchers a head start in the search for a melanosome transfer assay with said qualities to better understand pigment transfer.     

Fluorescence tools to investigate riboswitch structural dynamics

Riboswitches are novel regulatory elements that respond to cellular metabolites to control gene expression. They are constituted of highly conserved domains that have evolved to recognize specific metabolites. Such domains, so-called aptamers, are folded into intricate structures to enable metabolite recognition. Over the years, the development of ensemble and single-molecule fluorescence techniques has allowed to probe most of the mechanistic aspects of aptamer folding and ligand binding. In this review, we summarize the current fluorescence toolkit available to study riboswitch structural dynamics. We fist describe those methods based on fluorescent nucleotide analogues, mostly 2-aminopurine (2AP), to investigate short-range conformational changes, including some key steady-state and time-resolved examples that exemplify the versatility of fluorescent analogues as structural probes. The study of long-range structural changes by Förster resonance energy transfer (FRET) is mostly discussed in the context of single-molecule studies, including some recent developments based on the combination of single-molecule FRET techniques with controlled chemical denaturation methods. This article is part of a Special Issue entitled: Riboswitches.     

Unraveling secrets of telomeres: One molecule at a time

Telomeres play important roles in maintaining the stability of linear chromosomes. Telomere maintenance involves dynamic actions of multiple proteins interacting with long repetitive sequences and complex dynamic DNA structures, such as G-quadruplexes, T-loops and t-circles. Given the heterogeneity and complexity of telomeres, single-molecule approaches are essential to fully understand the structure–function relationships that govern telomere maintenance. In this review, we present a brief overview of the principles of single-molecule imaging and manipulation techniques. We then highlight results obtained from applying these single-molecule techniques for studying structure, dynamics and functions of G-quadruplexes, telomerase, and shelterin proteins.     

Single-molecule spectroscopic methods

Being praised for the mere fact of enabling the detection of individual fluorophores a dozen years ago, single-molecule techniques nowadays represent standard methods for the elucidation of the structural rearrangements of biologically relevant macromolecules. Single-molecule-sensitive techniques, such as fluorescence correlation spectroscopy, allow real-time access to a multitude of molecular parameters (e.g. diffusion coefficients, concentration and molecular interactions). As a result of various recent advances, this technique shows promise even for intracellular applications. Fluorescence imaging can reveal the spatial localization of fluorophores on nanometer length scales, whereas fluorescence resonance energy transfer supports a wide range of different applications, including real-time monitoring of conformational rearrangements (as in protein folding). Still in their infancy, single-molecule spectroscopic methods thus provide unprecedented insights into basic molecular mechanisms.     

Biophysical characterization of G-protein coupled receptor-peptide ligand binding

G-protein coupled receptors (GPCRs) are ubiquitous membrane proteins allowing intracellular responses to extracellular factors that range from photons of light to small molecules to proteins. Despite extensive exploitation of GPCRs as therapeutic targets, biophysical characterization of GPCR-ligand interactions remains challenging. In this minireview, we focus on techniques that have been successfully used for structural and biophysical characterization of peptide ligands binding to their cognate GPCRs. The techniques reviewed include solution-state nuclear magnetic resonance (NMR) spectroscopy, solid-state NMR, X-ray diffraction, fluorescence spectroscopy and single-molecule fluorescence methods, flow cytometry, surface plasmon resonance, isothermal titration calorimetry, and atomic force microscopy. The goal herein is to provide a cohesive starting point to allow selection of techniques appropriate to the elucidation of a given GPCR-peptide interaction.     

DNA and chromatin imaging with super-resolution fluorescence microscopy based on single-molecule localization

With the expansion of super-resolution fluorescence microscopy methods, it is now possible to access the organization of cells and materials at the nanoscale by optical means. This review discusses recent progress in super-resolution imaging of isolated and cell DNA using single-molecule localization methods. A high labeling density of photoswitchable fluorophores is crucial for these techniques, which can be provided by sequence independent DNA stains in which photoblinking reactions can be induced. In particular, unsymmetrical cyanine intercalating dyes in combination with special buffers can be used to image isolated DNA with a spatial resolution of 30-40 nm. For super-resolution imaging of chromatin, cell permeant cyanine dyes that bind the minor groove of DNA have the potential to become a useful alternative to the labeling of histones and other DNA-associated proteins. Other recent developments that are interesting in this context such as high density labeling methods or new DNA probes with photoswitching functionalities are also surveyed. Progress in labeling, optics, and single-molecule localization algorithms is being rapid, and it is likely to provide real insight into DNA structuring in cells and materials.     

smFRET studies of the ‘encounter’ complexes and subsequent intermediate states that regulate the selectivity of ligand binding

The selectivity with which a biomolecule can bind its cognate ligand when confronted by the vast array of structurally similar, competing ligands that are present in the cell underlies the fidelity of some of the most fundamental processes in biology. Because they collectively comprise one of only a few methods that can sensitively detect the ‘encounter’ complexes and subsequent intermediate states that regulate the selectivity of ligand binding, single-molecule fluorescence, and particularly single-molecule fluorescence resonance energy transfer (smFRET), approaches have revolutionized studies of ligand-binding reactions. Here, we describe a widely used smFRET strategy that enables investigations of a large variety of ligand-binding reactions, and discuss two such reactions, aminoacyl-tRNA selection during translation elongation and splice site selection during spliceosome assembly, that highlight both the successes and challenges of smFRET studies of ligand-binding reactions. We conclude by reviewing a number of emerging experimental and computational approaches that are expanding the capabilities of smFRET approaches for studies of ligand-binding reactions and that promise to reveal the mechanisms that control the selectivity of ligand binding with unprecedented resolution.     

A comparison of Förster resonance energy transfer analysis approaches for Nanodrop fluorometry

Ensemble Förster resonance energy transfer (FRET) results can be analyzed in a variety of ways. Due to experimental artifacts, the results obtained from different analysis approaches are not always the same. To determine the optimal analysis approach to use for Nanodrop fluorometry, we have performed both ensemble and single-molecule FRET studies on oligomers of double-stranded DNA. We compared the single-molecule FRET results with those obtained using various ensemble FRET analysis approaches. This comparison shows that for Nanodrop fluorometry, analyzing the increase of the acceptor fluorescence is less likely to introduce errors in estimates of FRET efficiencies compared with analyzing the fluorescence intensity of the donor in the absence and presence of the acceptor.