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Bacteriophage T7 single-stranded DNA-binding protein (gp2.5) binds to and protects transiently exposed regions of single-stranded DNA (ssDNA) while dynamically interacting with other proteins of the replication complex. We directly visualize fluorescently labelled T7 gp2.5 binding to ssDNA at the single-molecule level. Upon binding, T7 gp2.5 reduces the contour length of ssDNA by stacking nucleotides in a force-dependent manner, suggesting T7 gp2.5 suppresses the formation of secondary structure. Next, we investigate the binding dynamics of T7 gp2.5 and a deletion mutant lacking 21 C-terminal residues (gp2.5-Δ21C) under various template tensions. Our results show that the base sequence of the DNA molecule, ssDNA conformation induced by template tension, and the acidic terminal domain from T7 gp2.5 significantly impact on the DNA binding parameters of T7 gp2.5. Moreover, we uncover a unique template-catalyzed recycling behaviour of T7 gp2.5, resulting in an apparent cooperative binding to ssDNA, facilitating efficient spatial redistribution of T7 gp2.5 during the synthesis of successive Okazaki fragments. Overall, our findings reveal an efficient binding mechanism that prevents the formation of secondary structures by enabling T7 gp2.5 to rapidly rebind to nearby exposed ssDNA regions, during lagging strand DNA synthesis.

Fluorescence imaging in combination with single-particle tracking analysis has emerged as a powerful tool to study and characterize the motion of proteins moving in biological media. One of the main challenges in this approach is to reliably distinguish between directed and diffusive transport, especially for short and often noisy trajectories showing distinct, time- and place-dependent modes of motility. In this contribution, we present a windowed Mean-Square Displacements classifier (wMSDc) that is able to reliably (i) identify periods of diffusive and directed transport, (ii) extract position-dependent diffusion coefficients and velocities, and (iii) identify the location of switches in direction or motility mode in short (< 50 time points) and noisy single-molecule trajectories. We compare the performance of this approach to a Hidden Markov Model (HMM) method and a Moment Scaling Spectrum based method (DC-MSS) previously published and show that, in most cases, its performance is superior. We present a wide range of applications: from the movement of whole organisms and cells to protein-DNA interactionsin vitroand motor-protein dynamicsin vivo.Statement of SignificanceExtracting quantitative parameters from single-particle trajectories is a challenging task, especially in biological samples, which often show a high degree of heterogeneity. Trajectories can reveal switches between different types of motion; directed, diffusive and sub-diffusive motion. Usually, the length of these trajectories and their localization precision are limited by the experimental conditions. Here, we present a novel approach to analyse single molecule trajectories, windowed Mean-Square Displacement classifier (wMSDc) to reliably distinguish between directed and diffusive transport in the short trajectories with a finite precision of localization and integration time typically obtained when imaging single fluorescent proteins in living cells or organisms. We show that, using simulated and a wide range of experimental trajectories, wMSDc is a reliable method to extract motility parameters such as diffusion coefficient and velocity.

Recent advances in the design and measurement capabilities of optical tweezers instruments, and especially the combination with multi-color fluorescence detection, have accommodated a dramatic increase in the versatility of optical trapping. Quadruple (Q)-trap optical tweezers are an excellent example of such an advance, by providing three-dimensional control over two constructs and thereby enabling for example DNA-DNA braiding. However, the implementation of fluorescence detection in such a Q-trapping system poses several challenges: (1) since typical samples span a distance in the order of tens of micrometers, it requires imaging of a large field of view, (2) in order to capture fast molecular dynamics, fast imaging with single-molecule sensitivity is desired, (3) in order to study three-dimensional objects, it could be needed to detect emission light at different axial heights while keeping the objective lens and thus the optically trapped microspheres in a fixed position. In this chapter, we describe design guidelines for a fluorescence imaging module on a Q-trap system that overcomes these challenges and provide a step-by-step description for construction and alignment of such a system. Finally, we present detailed instructions for proof-of-concept experiments that can be used to validate and highlight the capabilities of the instruments.

Optical tweezers and fluorescence microscopy are powerful methods for investigating the mechanical and structural properties of biomolecules and for studying the dynamics of the biomolecular processes that these molecules are involved in. Here we provide an outline of the concurrent use of optical tweezers and fluorescence microscopy for analyzing biomolecular processes. In particular, we focus on the use of super-resolution microscopy in optical tweezers, which allows visualization of molecules at the higher molecular densities that are typically encountered in living systems. We provide specific details on the alignment procedures of the optical pathways for confocal fluorescence microscopy and 1D-STED microscopy and elaborate on how to diagnose and correct optical aberrations and STED phase plate misalignments.

Many genomic processes lead to the formation of underwound (negatively supercoiled) or overwound (positively supercoiled) DNA. These DNA topological changes regulate the interactions of DNA-binding proteins, including transcription factors, architectural proteins and topoisomerases. In order to advance our understanding of the structure and interactions of supercoiled DNA, we recently developed a single-molecule approach called Optical DNA Supercoiling (ODS). This method enables rapid generation of negatively supercoiled DNA (with between5% and 70% lower helical twist than nonsupercoiled DNA) using a standard dual-trap optical tweezers instrument. ODS is advantageous as it allows for combined force spectroscopy, fluorescence imaging, and spatial control of the supercoiled substrate, which is difficult to achieve with most other approaches. Here, we describe how to generate negatively supercoiled DNA using dual-trap optical tweezers. To this end, we provide detailed instructions on the design and preparation of suitable DNA substrates, as well as a step-by-step guide for how to control and calibrate the supercoiling density produced.

Optical tweezers are widely used to investigate biomolecules and biomolecular interactions. In these investigations, the biomolecules of interest are typically coupled to microscopic beads that can be optically trapped. Since high-intensity laser beams are required to trap such microscopic beads, laser-induced heating due to optical absorption is typically unavoidable. This chapter discusses how to identify, quantify, and control thermal effects in optical tweezers. We provide a brief overview of the reported causes and effects of unwanted heating in optical tweezers systems. Specific details are provided on methods to perform a temperature-independent trap calibration procedure. Finally, an effective temperature-control system is presented, and we discuss the operation of this system as well as the methods to measure the temperature at the optically trapped particle.

In preparation for mitotic cell division, the nuclear DNA of human cells is compacted into individualized, X-shaped chromosomes1. This metamorphosis is driven mainly by the combined action of condensins and topoisomerase IIα (TOP2A)2,3, and has been observed using microscopy for over a century. Nevertheless, very little is known about the structural organization of a mitotic chromosome. Here we introduce a workflow to interrogate the organization of human chromosomes based on optical trapping and manipulation. This allows high-resolution force measurements and fluorescence visualization of native metaphase chromosomes to be conducted under tightly controlled experimental conditions. We have used this method to extensively characterize chromosome mechanics and structure. Notably, we find that under increasing mechanical load, chromosomes exhibit nonlinear stiffening behaviour, distinct from that predicted by classical polymer models4. To explain this anomalous stiffening, we introduce a hierarchical worm-like chain model that describes the chromosome as a heterogeneous assembly of nonlinear worm-like chains. Moreover, through inducible degradation of TOP2A5 specifically in mitosis, we provide evidence that TOP2A has a role in the preservation of chromosome compaction. The methods described here open the door to a wide array of investigations into the structure and dynamics of both normal and disease-associated chromosomes.

In preparation for mitotic cell division, the nuclear DNA of human cells is compacted into individualized, X-shaped chromosomes

In the version of this article initially published, Extended Data Fig. 5 was a duplicate of Extended Data Fig. 6. The correct image is now in place in the HTML and PDF versions of the article.

Single molecule analysis , Single molecule analysis , کتابخانه مرکزی دانشگاه علوم پزشکی تهران

Transportation of organelles and biomolecules is vital for many cellular processes. Single-molecule (SM) fluorescence microscopy can expose molecular aspects of the dynamics that remain unresolved in ensemble experiments. For example, trajectories of individual, moving biomolecules can reveal velocity and changes therein, including pauses. We use SM imaging to study the dynamics of motor proteins and their cargo in the cilia of living C. elegans. To this end, we employ standard fluorescent proteins, an epi-illuminated, wide-field fluorescence microscope and mostly open-source software. This chapter describes the setup we use, the preparation of samples, a protocol for single-molecule imaging in C. elegans and data analysis.

One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow access to useful measurable parameters on time and length scales relevant for the biomolecular world. Before several detailed experimental approaches will be addressed, we will first give a general overview of single-molecule fluorescence microscopy. We start with discussing the phenomenon of fluorescence in general and the history of single-molecule fluorescence microscopy. Next, we will review fluorescent probes in more detail and the equipment required to visualize them on the single-molecule level. We will end with a description of parameters measurable with such approaches, ranging from protein counting and tracking, single-molecule localization super-resolution microscopy, to distance measurements with Förster Resonance Energy Transfer and orientation measurements with fluorescence polarization.

DNA metabolism and DNA compaction in vivo involve frequent interactions of remote DNA segments, mediated by proteins. In order to gain insight into such interactions, quadruple-trap optical tweezers have been developed. This technique provides an unprecedented degree of control through the ability to independently manipulate two DNA molecules in three dimensions. In this way, discrete regions of different DNA molecules can be brought into contact with one another, with a well-defined spatial configuration. At the same time, the tension and extension of the DNA molecules can be monitored. Furthermore, combining quadruple-trap optical tweezers with microfluidics makes fast buffer exchange possible, which is important for in situ generation of the dual DNA-protein constructs needed for these kinds of experiments. In this way, processes such as protein-mediated inter-DNA bridging can be studied with unprecedented control. This chapter provides a step-by-step description of how to perform a dual DNA manipulation experiment using combined quadruple-trap optical tweezers and microfluidics.

Optical manipulation techniques provide researchers the powerful ability to directly move, probe and interrogate molecular complexes. Quadruple optical trapping is an emerging method for optical manipulation and force spectroscopy that has found its primary use in studying dual DNA interactions, but is certainly not limited to DNA investigations. The key benefit of quadruple optical trapping is that two molecular strands can be manipulated independently and simultaneously. The molecular geometries of the strands can thus be controlled and their interactions can be quantified by force measurements. Accurate control of molecular geometry is of critical importance for the analysis of, for example, protein-mediated DNA-bridging, which plays an important role in DNA compaction. Here, we describe the design of a dedicated and robust quadruple optical trapping-instrument. This instrument can be switched straightforwardly to a high-resolution dual trap and it is integrated with microfluidics and single-molecule fluorescence microscopy, making it a highly versatile tool for correlative single-molecule analysis of a wide range of biomolecular systems.

Essential genomic transactions such as DNA-damage repair and DNA replication take place on single-stranded DNA (ssDNA) or require specific single-stranded/double-stranded DNA (ssDNA/dsDNA) junctions (SDSJ). A significant challenge in single-molecule studies of DNA-protein interactions using optical trapping is the design and generation of appropriate DNA templates. In contrast to dsDNA, only a limited toolbox is available for the generation of ssDNA constructs for optical tweezers experiments. Here, we present several kinds of DNA templates suitable for single-molecule experiments requiring segments of ssDNA of several kilobases in length. These different biotinylated dsDNA templates can be tethered between optically trapped microspheres and can, by the subsequent use of force-induced DNA melting, be converted into partial or complete ssDNA molecules. We systematically investigated the time scale and efficiency of force-induced melting at different ionic strengths for DNA molecules of different sequences and lengths. Furthermore, we quantified the impact of microspheres of different sizes on the lifetime of ssDNA tethers in optical tweezers experiments. Together, these experiments provide deeper insights into the variables that impact the production of ssDNA for single molecules studies and represent a starting point for further optimization of DNA templates that permit the investigation of protein binding and kinetics on ssDNA.

Kinesin is an essential eukaryotic protein that drives intracellular transport of cargo, such as vesicles and organelles. It is the smallest motor protein known that converts free energy obtained from ATP hydrolysis into mechanical work, by stepping along microtubules. The enzymatic cycle of kinesin is tightly coupled to mechanical action. How kinesin's two identical motor domains (that both bind and hydrolyze ATP and bind to a microtubule) bring about motility has been the subject of much research. Recently, we have developed and applied a single-motor motility assay based on confocal fluorescence microscopy to measure changes in distance and orientation of the two motor domains during processive walking using Förster resonance energy transfer. The key benefit of this approach is its unprecedented time resolution of about 0.1 ms. In this chapter, we explain our approach in detailed protocols.

The technically challenging field of single-molecule biophysics has established itself in the last decade by granting access to detailed information about the fate of individual biomolecules, unattainable in traditional biochemical assays. The appeal of single-molecule methods lies in the directness of the information obtained from individual biomolecules. Technological improvements in single-molecule methods have made it possible to combine optical tweezers, fluorescence microscopy, and microfluidic flow systems. Such a combination of techniques has opened new possibilities to study complex biochemical reactions on the single-molecule level. In this chapter, we provide general considerations for the development of a combined optical trapping, fluorescence microscopy, and microfluidics instrument, along with methods to solve technical issues that are critical for designing successful experiments. Finally, we present several experiments to illustrate the power of this combination of techniques.

To obtain high-resolution information on position or conformation of a molecule and at the same time apply forces to it, one can combine optical trapping with single-molecule fluorescence microscopy. The technical challenge in such an experiment is to discriminate a minute fluorescence signal from the much larger background signals caused by the trap and the fluorescence excitation laser light. We show here that this is feasible even when the fluorophore is directly attached to the trapped particle, by using optimized optical filters. We found, however, that the photostability of the fluorophores we tested suffered from the presence of the additional laser light used for trapping. We found that bleaching rates increased linearly with both the intensity of the trapping laser and the intensity of the fluorescence excitation light. Photobleaching rates were unaffected by the presence or absence of oxygen, but were significantly diminished in the presence of antioxidants. Our results indicate that the enhanced photobleaching is caused by the absorption of a visible photon followed by the excited-state absorption of a near-infrared photon. The higher excited singlet states generated in this way readily form nonfluorescent dye cations. We found that different dyes suffer to a different extent from the excited-state absorption, with Cy3 being worst and tetramethylrhodamine least affected.