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The hybridization kinetic of an oligonucleotide to its template is a fundamental step in many biological processes such as replication arrest, CRISPR recognition, DNA sequencing, DNA origami, etc. Although single kinetic descriptions exist for special cases of this problem, there are no simple general prediction schemes. In this work, we have measured experimentally, with no fluorescent labelling, the displacement of an oligonucleotide from its substrate in two situations: one corresponding to oligonucleotide binding/unbinding on ssDNA and one in which the oligonucleotide is displaced by the refolding of a dsDNA fork. In this second situation, the fork is expelling the oligonucleotide thus significantly reducing its residence time. To account for our data in these two situations, we have constructed a mathematical model, based on the known nearest neighbour dinucleotide free energies, and provided a good estimate of the residence times of different oligonucleotides (DNA, RNA, LNA) of various lengths in different experimental conditions (force, temperature, buffer conditions, presence of mismatches, etc.). This study provides a foundation for the dynamics of oligonucleotide displacement, a process of importance in numerous biological and bioengineering contexts.

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Accurate decoding of nucleic acid variation is critical to understand the complexity and regulation of genome function. Here we use a single-molecule magnetic tweezer (MT) platform to identify sequence variation and map a range of important epigenetic base modifications with high sensitivity, specificity, and precision in the same single molecules of DNA or RNA. We have also developed a highly specific amplification-free CRISPR-Cas enrichment strategy to isolate genomic regions from native DNA. We demonstrate enrichment of DNA from both E. coli and the FMR1 5’UTR coming from cells derived from a Fragile X carrier. From these kilobase-length enriched molecules we could characterize the differential levels of adenine and cytosine base modifications on E. coli, and the repeat expansion length and methylation status of FMR1. Together these results demonstrate that our platform can detect a variety of genetic, epigenetic, and base modification changes concomitantly within the same single molecules., Wang et al. show how genetic sequence and base modifications can be detected simultaneously on single molecules of both DNA and RNA using magnetic tweezers. They also demonstrate an amplification-free CRISPR/Cas9-based strategy for isolating target regions from native DNA and apply this approach to the isolation of targets from E. coli and human genomic DNA.

Surface colonization underpins microbial ecology on terrestrial environments. Although factors that mediate bacteria–substrate adhesion have been extensively studied, their spatiotemporal dynamics during the establishment of microcolonies remains largely unexplored. Here, we use laser ablation and force microscopy to monitor single-cell adhesion during the course of microcolony formation. We find that adhesion forces of the rod-shaped bacteria Escherichia coli and Pseudomonas aeruginosa are polar. This asymmetry induces mechanical tension, and drives daughter cell rearrangements, which eventually determine the shape of the microcolonies. Informed by experimental data, we develop a quantitative model of microcolony morphogenesis that enables the prediction of bacterial adhesion strength from simple time-lapse measurements. Our results demonstrate how patterns of surface colonization derive from the spatial distribution of adhesive factors on the cell envelope., It is unclear how cell adhesion and elongation coordinate during formation of bacterial microcolonies. Here, Duvernoy et al. monitor microcolony formation in rod-shaped bacteria, and show that patterns of surface colonization derive from the spatial distribution of adhesive factors on the cell envelope.

The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. Whereas many of these approaches use fusion between a light-activable protein and the protein of interest to control the activity of the latter, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly, and locally in a live organism. We present that approach and its uses in a variety of physiological contexts.

Helicases are a broad family of enzymes that separate nucleic acid double strand structures (DNA/DNA, DNA/RNA or RNA/RNA) and thus are essential to DNA replication and the maintenance of nucleic acid integrity. We review the picture that has emerged from single molecule studies of the mechanisms of DNA and RNA helicases and their interactions with other proteins. Many features have been uncovered by these studies that were obscured by bulk studies, such as DNA strands switching, mechanical (rather than biochemical) coupling between helicases and polymerases, helicase-induced re-hybridization and stalled fork rescue. This article is protected by copyright. All rights reserved.

The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. While many of these approaches use a fusion between a light activatable protein and the protein of interest to control the activity of the latter, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly and locally in a live organism. Here, we present the experimental details behind this approach.

A quantitative description of the molecular networks that sustain morphogenesis is one of the main challenges of developmental biology. In particular, a molecular understanding of the segmentation of the antero-posterior axis in vertebrates has yet to be achieved. This process known as somitogenesis is believed to result from the interactions between a well-studied genetic oscillator and a less established posterior-moving determination wavefront. Here we describe a molecular model for somitogenesis that couples a moving morphogen wavefront with the somitogenetic oscillator. The wavefront is due to a switch between stable states that results from reciprocal negative feedbacks of Retinoic Acid (RA) on the activation of a kinase ErK and of ErK on RA synthesis. We suggest a molecular mechanism by which that switch can be triggered by the somitogenetic clock. The model quantitatively accounts for the shortening of the pre-somitic mesoderm (PSM) in zebrafish in response to the decrease during somitogenesis in the concentration of a morphogen (Fgf8). The generality and robustness of the model allows for its validation (or invalidation) in other model organisms.

The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. While many of these approaches use a fusion between a light activatable protein and the protein of interest to control the activity of the latter, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly and locally in a live organism. Here, we present the experimental details behind this approach.

This chapter reviews the use of fluorescent methods in the study of single molecules, how the foundations of fluorescence are rooted in Einstein’s description of absorption and emission (spontaneous and stimulated), and their quantum-mechanical explanation in terms of transitions between quantized energy levels, as represented in Jablonski diagrams. It describes the non-radiative channels which compete with fluorescent emission, decrease its efficiency, and ultimately destroy the fluorescent molecule. Fluorescence Polarization Spectroscopy, FRET, and FCS are briefly presented. Without reviewing the various available fluorophores, it describes the various illumination methods used to study them, sketching super-resolution methods (STED, STORM, PALM) that have recently allowed fluorophores to be resolved to a few tens of nanometres. Finally, it describes the considerations (bandwidth, signal to noise, signal to background) used in choosing a single-molecule fluorescence detector, and the extraction of the diffusion constant of a fluorophore from the finite time, noisy traces of its positions.

This chapter provides a quick introduction to the structural properties of nucleic acids (DNA and RNA). It describes the famed double-helical structure of DNA, the more complex 3D structures adopted by RNA, and the random (possibly) twisted coil that nucleic acid can display at large scales.

This chapter discusses single-molecule approaches in the study of topoisomerases. After introducing the problem posed by DNA entanglement, it describes type I and type II topoisomerases, which solve that issue. Single-molecule assays have nailed down the different mechanisms of bacterial and eukaryotic type I topoisomerases. The properties of type II topoisomerases are then described. Single-molecule experiments have shown that they relax DNA torsion by two units, passing one dsDNA segment through a break in another segment. However, while topoII relaxes positive and negative supercoils similarly, topoIV relaxes positive supercoils quickly and processively, but negative ones slowly and distributively. This chiral discrimination is compared with the activity of the enzyme on two catenated DNA molecules. After describing single-molecule assays of the activity of gyrases, in-vivo investigations of single fluorescently labelled topoIV units in single E.coli are discussed, with concluding remarks on the future of single-molecule DNA/protein studies.

Single molecule methods have revolutionized the field of Biophysics. Much of the current applications of these methods are devoted to in vitro studies. It is our hope that the knowledge gained from these studies and described in this book will open a new vista on the in vivo investigation of cellular and physiological processes.

This book presents a comprehensive overview of the foundations of single-molecule studies, based on manipulation of the molecules and observation of these with fluorescent probes. It first discusses the forces present at the single-molecule scale, the methods to manipulate them, and their pros and cons. It goes on to present an introduction to single-molecule fluorescent studies based on a quantum description of absorption and emission of radiation due to Einstein. Various considerations in the study of single molecules are introduced (including signal to noise, non-radiative decay, triplet states, etc.) and some novel super-resolution methods are sketched. The elastic and dynamic properties of polymers, their relation to experiments on DNA and RNA, and the structural transitions observed in those molecules upon stretching, twisting, and unzipping are presented. The use of these single-molecule approaches for the investigation of DNA–protein interactions is highlighted via the study of DNA and RNA polymerases, helicases, and topoisomerases. Beyond the confirmation of expected mechanisms (e.g., the relaxation of DNA torsion by topoisomerases in quantized steps) and the discovery of unexpected ones (e.g., strand-switching by helicases, DNA scrunching by RNA polymerases, and chiral discrimination by bacterial topoII), these approaches have also fostered novel (third generation) sequencing technologies.

This chapter discusses the application of single-molecule approaches in the study of DNA and RNA polymerases. After an introduction to DNA replication and the structure of DNA polymerases, it reviews experiments on DNA polymerization on stretched ssDNA, moving on to DNA polymerization at a stretched DNA fork (mimicking the replication fork). Next it looks at single-molecule sequencing approaches based on DNA polymerization with sequential incorporation of fluorescently labelled nucleotides, comparing with nanopore sequencing. It outlines the use of fluorescent approaches in the study of replication dynamics in vivo in single cells, then discussing transcription by RNA polymerases, the stages of transcription (open-complex, abortive initiation, transcription elongation, termination), and the general structure of RNA polymerases. It describes single-molecule experiments (using manipulation/fluorescent approaches) of the transcription stages and ends with a discussion of experiments studying the dynamics of transcription in vivo at a single locus in a eukaryotic cell with fluorescent labelling.

This chapter reviews models which describe the elastic properties of stretched polymers—the Kratky–Porod, Freely Jointed Chain (FJC), and Worm-Like Chain (WLC) models—and the effect of self-avoidance on results derived from these. The models are compared with double-stranded DNA (dsDNA) stretching experiments. Dynamics of a single polymer in the presence (Zimm model) or absence (Rouse model) of hydrodynamic interactions between its segments is described, and results on the dynamics of dsDNA and ssDNA of various lengths are discussed. Theoretical and experimental behaviour of twisted DNA is described, deducing the molecule’s torsional modulus and its coupling between stretching and twisting. After discussing the braiding of two DNA molecules and simulation of the twisting and stretching of DNA molecules, this chapter describes the results of stretching experiments on ssDNA and RNA, where self-avoiding and base-pairing interactions contribute to elastic behaviour.

This chapter describes the various methods used to manipulate single DNA molecules and the considerations in the choice of one particular method. It starts with a description of DNA end-labelling, necessary to anchor the molecule to surfaces or beads that can be manipulated. A particular application of DNA anchoring is molecular combing, whereby the molecule is stretched on a surface by a receding meniscus. DNA rearrangements and replication bubbles can then be observed by fluorescence on these straightened molecules. It then looks at the forces at the molecular scale, which range from the smallest one due to thermal agitation, to the largest associated with breaking a covalent bond, via entropic and non-covalent bonding forces. It describes the tools used to manipulate single molecules (micro-needles, AFM cantilevers, optical, magnetic, and acoustic tweezers and traps, etc.), comparing their performances in terms of bandwidth and signal to noise (i.e., force and extension resolutions).

In this chapter we discuss the various structural transitions observed on dsDNA upon twisting and stretching: the transition to denatured DNA at negative twist and to P-DNA at positive twist; the transition to S-DNA at large force and its relation with DNA melting. We discuss mechanical unzipping of DNA and show how DNA rehybridization under tension in the presence of complementary oligonucleotides can be used to sequence the molecule.

Helicases are a broad family of enzymes that perform crucial functions in DNA replication and in the maintenance of DNA and RNA integrity. A detailed mechanical study of helicases on DNA and RNA is possible using single molecule manipulation methods. Among those, magnetic tweezers (or traps) present a convenient, moderate throughput assay (tens of enzymes can be monitored simultaneously) that allow for high resolution (single base-pair) studies of these enzymes in various conditions and on various substrates (double and single stranded DNA and RNA). Here we discuss various implementation of the basic assay relevant for these studies.

A quantitative description of the molecular networks that sustain morphogenesis is one of the challenges of developmental biology. Specifically, a molecular understanding of the segmentation of the antero-posterior axis in vertebrates has yet to be achieved. This process known as somitogenesis is believed to result from the interactions between a genetic oscillator and a posterior-moving determination wavefront. Here we quantitatively study and perturb the network in zebrafish that sustains this wavefront and compare our observations to a model whereby the wavefront is due to a switch between stable states resulting from reciprocal negative feedbacks of Retinoic Acid (RA) on the activation of ERK and of ERK on RA synthesis. This model quantitatively accounts for the near linear shortening of the post-somitic mesoderm (PSM) in response to the observed exponential decrease during somitogenesis of the mRNA concentration of a morphogen (Fgf8). It also accounts for the observed dynamics of the PSM when the molecular components of the network are perturbed. The generality of our model and its robustness allows for its test in other model organisms.

For over a century, the resolution of microscopes has been limited by the wavelength of the light emitted by the sample, a limit known as Abbe's limit. During the past 10 years, a number of imaging techniques, reviewed in this chapter, have allowed this apparent limitation to be overcome, to obtain microscopic images with essentially unlimited resolution.

Homeoproteins of the Engrailed family are involved in the patterning of mesencephalic boundaries through a mechanism classically ascribed to their transcriptional functions. In light of recent reports on the paracrine activity of homeoproteins, including Engrailed, we asked whether Engrailed intercellular transfer was also involved in brain patterning and boundary formation. Using time-controlled activation of Engrailed combined with tools that block its transfer, we show that the positioning of the diencephalic-mesencephalic boundary (DMB) requires Engrailed paracrine activity. Both zebrafish Eng2a and Eng2b are competent for intercellular transfer in vivo, but only extracellular endogenous Eng2b, and not Eng2a, participates in DMB positioning. In addition, disruption of the Pbx-interacting motif in Engrailed, known to strongly reduce the gain-of-function phenotype, also downregulates Engrailed transfer, thus revealing an unsuspected participation of the Pbx interaction domain in this pathway., SUMMARY: The paracrine activity of Engrailed, together with its PBX-interacting hexapeptide motif, is involved in boundary formation during brain development in zebrafish.

The small and synthetically easily accessible 7-diethylamino-4-thiocoumarinylmethyl photolabile protecting group has been validated for uncaging with blue light. It exhibits a significant action cross-section for uncaging in the 470-500 nm wavelength range and a low light absorption between 350 and 400 nm. These attractive features have been implemented in living zebrafish embryos to perform chromatic orthogonal photoactivation of two biologically active species controlling biological development with UV and blue-cyan light sources, respectively.

All-trans retinoic acid (RA) is a key player in many developmental pathways. Most methods used to study its effects in development involve continuous all-trans RA activation by incubation in a solution of all-trans RA or by implanting all-trans RA-soaked beads at desired locations in the embryo. Here we show that the UV-driven photo-isomerization of 13-cis RA to the trans-isomer (and vice versa) can be used to non-invasively and quantitatively control the concentration of all-trans RA in a developing embryo in time and space. This facilitates the global or local perturbation of developmental pathways with a pulse of all-trans RA of known concentration or its inactivation by UV illumination. In zebrafish embryos in which endogenous synthesis of all-trans RA is impaired, incubation for as little as 5 minutes in 1 nM all-trans RA (a pulse) or 5 nM 13-cis RA followed by 1-minute UV illumination is sufficient to rescue the development of the hindbrain if performed no later than bud stage. However, if subsequent to this all-trans RA pulse the embryo is illuminated (no later than bud stage) for 1 minute with UV light (to isomerize, i.e. deactivate, all-trans RA), the rescue of hindbrain development is impaired. This suggests that all-trans RA is sequestered in embryos that have been transiently exposed to it. Using 13-cis RA isomerization with UV light, we further show that local illumination at bud stage of the head region (but not the tail) is sufficient to rescue hindbrain formation in embryos whose all-trans RA synthetic pathway has been impaired.

Through its capability to transiently pack and unpack our genome, chromatin is a key player in the regulation of gene expression. Single-molecule approaches have recently complemented conventional biochemical and biophysical techniques to decipher the complex mechanisms ruling chromatin dynamics. Micromanipulations with tweezers (magnetic or optical) and imaging with molecular microscopy (electron or atomic force) have indeed provided opportunities to handle and visualize single molecules, and to measure the forces and torques produced by molecular motors, along with their effects on DNA or nucleosomal templates. By giving access to dynamic events that tend to be blurred in traditional biochemical bulk experiments, these techniques provide critical information regarding the mechanisms underlying the regulation of gene activation and deactivation by nucleosome and chromatin structural changes. This minireview describes some single-molecule approaches to the study of ATP-consuming molecular motors acting on DNA, with applications to the case of nucleosome-remodelling machines.

Magnetic traps provide a simple technique to pull and twist a variety of biomolecules and monitor the resulting change in extension. They have been used with great success to investigate the interaction of stretched and supercoiled DNA and DNA fibers (e.g. chromatin) with a great variety of enzymes. In this small review we will address their recent use in the study of topoisomerases, gyrase, DNA translocases and various structural proteins.

Topoisomerase IV (Topo IV), an essential ATP-dependent bacterial type II topoisomerase, transports one segment of DNA through a transient double-strand break in a second segment of DNA. In vivo, Topo IV unlinks catenated chromosomes before cell division and relaxes positive supercoils generated during DNA replication. In vitro, Topo IV relaxes positive supercoils at least 20-fold faster than negative supercoils. The mechanisms underlying this chiral discrimination by Topo IV and other type II topoisomerases remain speculative. We used magnetic tweezers to measure the relaxation rates of single and multiple DNA crossings by Topo IV. These measurements allowed us to determine unambiguously the relative importance of DNA crossing geometry and enzymatic processivity in chiral discrimination by Topo IV. Our results indicate that Topo IV binds and passes DNA strands juxtaposed in a nearly perpendicular orientation and that relaxation of negative supercoiled DNA is perfectly distributive. Together, these results suggest that chiral discrimination arises primarily from dramatic differences in the processivity of relaxing positive and negative supercoiled DNA: Topo IV is highly processive on positively supercoiled DNA, whereas it is perfectly distributive on negatively supercoiled DNA. These results provide fresh insight into topoisomerase mechanisms and lead to a model that reconciles contradictory aspects of previous findings while providing a framework to interpret future results.

Driven by a deluge of data, biology is undergoing a transition to a more quantitative science. Making sense of the data, building new models, asking the right questions and designing smart experiments to answer them are becoming ever more relevant. In this endeavour, nonlinear approaches can play a fundamental role. The biochemical reactions that underlie life are very often nonlinear. The functional features exhibited by biological systems at all levels (from the activity of an enzyme to the organization of a colony of ants, via the development of an organism or a functional module like the one responsible for chemotaxis in bacteria) are dynamically robust. They are often unaffected by order of magnitude variations in the dynamical parameters, in the number or concentrations of actors (molecules, cells, organisms) or external inputs (food, temperature, pH, etc). This type of structural robustness is also a common feature of nonlinear systems, exemplified by the fundamental role played by dynamical fixed points and attractors and by the use of generic equations (logistic map, Fisher–Kolmogorov equation, the Stefan problem, etc.) in the study of a plethora of nonlinear phenomena. However, biological systems differ from these examples in two important ways: the intrinsic stochasticity arising from the often very small number of actors and the role played by evolution. On an evolutionary time scale, nothing in biology is frozen. The systems observed today have evolved from solutions adopted in the past and they will have to adapt in response to future conditions. The evolvability of biological system uniquely characterizes them and is central to biology. As the great biologist T Dobzhansky once wrote: 'nothing in biology makes sense except in the light of evolution'.

By monitoring the response of individual protein and DNA molecules to pulling and twisting, biophysicists can learn much about their structure and their interactions. In this review we aim to give a flavour of the kinds of investigations that single-molecule techniques have made possible.

The Snf2 family represents a functionally diverse class of ATPase sharing the ability to modify DNA structure. Here, we use a magnetic trap and an atomic force microscope to monitor the activity of a member of this class: the RSC complex. This enzyme caused transient shortenings in DNA length involving translocation of typically 400 bp within 2 s, resulting in the formation of a loop whose size depended on both the force applied to the DNA and the ATP concentration. The majority of loops then decrease in size within a time similar to that with which they are formed, suggesting that the motor has the ability to reverse its direction. Loop formation was also associated with the generation of negative DNA supercoils. These observations support the idea that the ATPase motors of the Snf2 family of proteins act as DNA translocases specialized to generate transient distortions in DNA structure.

DNA encounters topological problems in vivo because of its extended double-helical structure. As a consequence, the semiconservative mechanism of DNA replication leads to the formation of DNA braids or catenanes, which have to be removed for the completion of cell division. To get a better understanding of these structures, we have studied the elastic behavior of two braided nicked DNA molecules using a magnetic trap apparatus. The experimental data let us identify and characterize three regimes of braiding: a slightly twisted regime before the formation of the first crossing, followed by genuine braids which, at large braiding number, buckle to form plectonemes. Two different approaches support and quantify this characterization of the data. First, Monte Carlo (MC) simulations of braided DNAs yield a full description of the molecules' behavior and their buckling transition. Second, modeling the braids as a twisted swing provides a good approximation of the elastic response of the molecules as they are intertwined. Comparisons of the experiments and the MC simulations with this analytical model allow for a measurement of the diameter of the braids and its dependence upon entropic and electrostatic repulsive interactions. The MC simulations allow for an estimate of the effective torsional constant of the braids (at a stretching force F = 2 pN): C(b) approximately 48 nm (as compared with C approximately 100 nm for a single unnicked DNA). Finally, at low salt concentrations and for sufficiently large number of braids, the diameter of the braided molecules is observed to collapse to that of double-stranded DNA. We suggest that this collapse is due to the partial melting and fraying of the two nicked molecules and the subsequent right- or left-handed intertwining of the stretched single strands.

The overall topology of DNA profoundly influences the regulation of transcription and is determined by DNA flexibility as well as the binding of proteins that induce DNA torsion, distortion, and/or looping. Gal repressor (GalR) is thought to repress transcription from the two promoters of the gal operon of Escherichia coli by forming a DNA loop of ≈40 nm of DNA that encompasses the promoters. Associated evidence of a topological regulatory mechanism of the transcription repression is the requirement for a supercoiled DNA template and the histone-like heat unstable nucleoid protein (HU). By using single-molecule manipulations to generate and finely tune tension in DNA molecules, we directly detected GalR/HU-mediated DNA looping and characterized its kinetics, thermodynamics, and supercoiling dependence. The factors required for gal DNA looping in single-molecule experiments (HU, GalR and DNA supercoiling) correspond exactly to those necessary for gal repression observed both in vitro and in vivo . Our single-molecule experiments revealed that negatively supercoiled DNA, under slight tension, denatured to facilitate GalR/HU-mediated DNA loop formation. Such topological intermediates may operate similarly in other multiprotein complexes of transcription, replication, and recombination.

Type-II topoisomerases are responsible for untangling DNA during replication by removing supercoiled and interlinked DNA structures. Using a single-molecule micromanipulation setup, we follow the real-time decatenation of two mechanically braided DNA molecules by Drosophila melanogaster topoisomerase (Topo) II and Escherichia coli Topo IV. Although Topo II relaxes left-handed (L) and right-handed (R-) braids similarly at a rate of ≈2.9 s – 1 , Topo IV has a marked preference for L-braids, which it relaxes completely and processively at a rate of ≈2.4 s – 1 . However, Topo IV can unlink R-braids at about half that rate when they supercoil to form L-plectonemes. These results imply that the preferred substrate for unlinking by Topo IV has the symmetry of an L-crossing and shed new light on the decatenation of daughter strands during DNA replication, which are usually assumed to be linked in an R-braid.

In this paper we review the biophysics revealed by stretching single biopolymers. During the last decade various techniques have emerged allowing micromanipulation of single molecules and simultaneous measurements of their elasticity. Using such techniques, it has been possible to investigate some of the interactions playing a role in biology. We shall first review the simplest case of a non-interacting polymer and then present the structural transitions in DNA, RNA and proteins that have been studied by single-molecule techniques. We shall explain how these techniques permit a new approach to the protein folding/unfolding transition.