Unfolding rates of 1:1 and 2:1 complex of CX-5461 and c-MYC promoter G-quadruplexes revealed by single-molecule force spectroscopy.

CX-5461, also known as pidnarulex, is a strong G4 stabilizer and has received FDA fast-track designation for BRCA1- and BRCA2- mutated cancers. However, quantitative measurements of the unfolding rates of CX-5461-G4 complexes which are important for the regulation function of G4s, remain lacking. Here, we employ single-molecule magnetic tweezers to measure the unfolding force distributions of c-MYC G4s in the presence of different concentrations of CX-5461. The unfolding force distributions exhibit three discrete levels of unfolding force peaks, corresponding to three binding modes. In combination with a fluorescent quenching assay and molecular docking to previously reported ligand-c-MYC G4 structure, we assigned the ~69 pN peak corresponding to the 1:1 (ligand:G4) complex where CX-5461 binds at the G4's 5'-end. The ~84 pN peak is attributed to the 2:1 complex where CX-5461 occupies both the 5' and 3'. Furthermore, using the Bell-Arrhenius model to fit the unfolding force distributions, we determined the zero-force unfolding rates of 1:1, and 2:1 complexes to be (2.4 ± 0.9) × 10-8 s-1 and (1.4 ± 1.0) × 10-9 s-1 respectively. These findings provide valuable insights for the development of G4-targeted ligands to combat c-MYC-driven cancers.

Unravelling DNA Organization with Single-Molecule Force Spectroscopy Using Magnetic Tweezers.

Genomes carry the genetic blueprint of all living organisms. Their organization requires strong condensation as well as carefully regulated accessibility to specific genes for proper functioning of their hosts. The study of the structure and dynamics of the proteins that organize the genome has benefited tremendously from the development of single-molecule force spectroscopy techniques that allow for real-time, nanometer accuracy measurements of the compaction of DNA and manipulation with pico-Newton scale forces. Magnetic tweezers, in particular, have the unique ability to complement such force spectroscopy with the control over the linking number of the DNA molecule, which plays an important role when DNA-organizing proteins form or release wraps, loops, and bends in DNA. Here, we describe all the necessary steps to prepare DNA substrates for magnetic tweezers experiments, assemble flow cells, tether DNA to a magnetic bead inside a flow cell, and manipulate and record the extension of such DNA tethers. Furthermore, we explain how mechanical parameters of nucleoprotein filaments can be extracted from the data.

Force-regulated chaperone activity of BiP/ERdj3 is opposite to their homologs DnaK/DnaJ.

Polypeptide chains experience mechanical tension while translocating through cellular tunnels, which are subsequently folded by molecular chaperones. However, interactions between tunnel-associated chaperones and these emerging polypeptides under force is not completely understood. Our investigation focused on mechanical chaperone activity of two tunnel-associated chaperones, BiP and ERdj3 both with and without mechanical constraints and comparing them with their cytoplasmic homologs: DnaK and DnaJ. While BiP/ERdj3 have been observed to exhibit robust foldase activity under force, DnaK/DnaJ showed holdase function. Importantly, the tunnel-associated chaperones (BiP/ERdj3) transitioned to a holdase state in the absence of force, indicating a force-dependent chaperone behavior. This chaperone-driven folding event in the tunnel generated an additional mechanical energy of up to 54 zJ, potentially aiding protein translocation. Our findings align with strain theory, where chaperones with higher intrinsic deformability act as mechanical foldases (BiP, ERdj3), while those with lower deformability serve as holdases (DnaK and DnaJ). This study thus elucidates the differential mechanically regulated chaperoning activity and introduces a novel perspective on co-translocational protein folding.

Horizontal magnetic tweezers and its applications in single molecule micromanipulation experiments.

Magnetic tweezers (MTs) have become indispensable tools for gaining mechanistic insights into the behavior of DNA-processing enzymes and acquiring detailed, high-resolution data on the mechanical properties of DNA. Currently, MTs have two distinct designs: vertical and horizontal (or transverse) configurations. While the vertical design and its applications have been extensively documented, there is a noticeable gap in comprehensive information pertaining to the design details, experimental procedures, and types of studies conducted with horizontal MTs. This article aims to address this gap by providing a concise overview of the fundamental principles underlying transverse MTs. It will explore the multifaceted applications of this technique as an exceptional instrument for scrutinizing DNA and its interactions with DNA-binding proteins at the single-molecule level.

Observing G4 formation and its resolution by Pif1 in real time by manipulation under magnetic tweezers.

G-quadruplexes (G4s) are nucleic acids secondary structures that may form in guanine-rich sequences, either intra or inter-molecularly. Ability of a primary sequence to form a G4 can be predicted computationally with an improving accuracy as well as tested in bulk using biophysical measurements. As a result, G4 density maps have been devised for a large number of genomes from all life kingdoms. Experimental validation of the formation of G4s in vivo however remains indirect and relies on their stabilization with small molecules, antibodies or proteins, or mutational studies, in order to measure downstream effects on gene expression or genome stability for example. Although numerous techniques exist to observe spontaneous formation of G4s in single-stranded DNA, observing G4 formation in double-stranded DNA (dsDNA) is more challenging. However, it is particularly relevant to understand if a given G4 sequence forms stably in a dsDNA context, if it is stable enough to dock proteins or pose a challenge to molecular motors such as helicases or polymerases. In essence, G4s can be a threat to genomic stability but carry as well as the potential to be elements of a structural language in the non-replicating genome. To study quantitatively the formation dynamics and stability of single intramolecular G4s embedded in dsDNA, we have adapted techniques of DNA manipulation under magnetic tweezers. This technique also allows to study encounters of molecular motors with G4 at a single molecule resolution, in order to gain insight into the specificity of G4 resolution by molecular motors, and its efficiency. The procedures described here include the design of the G4 substrate, the study of G4 formation probability and lifetime in dsDNA, as well as procedures to characterize the encounter between the Pif1 helicase and a G4 until G4 resolution. The procedures that we described here can easily be extended to the study of other G4s or molecular motors.

High-speed measurements of SNARE-complexin interactions using magnetic tweezers.

In neuroscience, understanding the mechanics of synapses, especially the function of force-sensitive proteins at the molecular level, is essential. This need emphasizes the importance of precise measurement of synaptic protein interactions. Addressing this, we introduce high-resolution magnetic tweezers (MT) as a novel method to probe the mechanics of synapse-related proteins with high precision. We demonstrate this technique through studying SNARE-complexin interactions, crucial for synaptic transmission, showcasing its capability to apply specific forces to individual molecules. Our results reveal that high-resolution MT provides in-depth insights into the stability and dynamic transitions of synaptic protein complexes. This method is a significant advancement in synapse biology, offering a new tool for researchers to investigate the impact of mechanical forces on synaptic functions and their implications for neurological disorders.

Integrating magnetic tweezers and single-molecule FRET: A comprehensive approach to studying local and global conformational changes simultaneously.

This chapter presents the integration of magnetic tweezers with single-molecule FRET technology, a significant advancement in the study of nucleic acids and other biological systems. We detail the technical aspects, challenges, and current status of this hybrid technique, which combines the global manipulation and observation capabilities of magnetic tweezers with the local conformational detection of smFRET. This innovative approach enhances our ability to analyze and understand the molecular mechanics of biological systems. The chapter serves as our first formal documentation of this method, offering insights and methodologies developed in our laboratory over the past decade.

Magnetic tweezers principles and promises.

Magnetic tweezers have become popular with the outbreak of single molecule micromanipulation: catching a single molecule of DNA, RNA or a single protein and applying mechanical constrains using micron-size magnetic beads and magnets turn out to be easy. Various factors have made this possible: the fact that manufacturers have been preparing these beads to catch various biological entities-the ease of use provided by magnets which apply a force or a torque at a distance thus inside a flow cell-some chance: since the forces so generated are in the right range to stretch a single molecule. This is a little less true for torque. Finally, one feature which also appears very important is the simplicity of their calibration using Brownian motion. Here we start by describing magnetic tweezers used routinely in our laboratory where we have tried to develop a device as simple as possible so that the experimentalist can really focus on the biological aspect of the biomolecules that he/she is interested in. We discuss the implications of the various components and their important features. Next, we summarize what is easy to achieve and what is less easy. Then we refer to contributions by other groups who have brought valuable insights to improve magnetic tweezers.

Exploring the free energy landscape of proteins using magnetic tweezers.

Proteins fold to their native states by searching through the free energy landscapes. As single-domain proteins are the basic building block of multiple-domain proteins or protein complexes composed of subunits, the free energy landscapes of single-domain proteins are of critical importance to understand the folding and unfolding processes of proteins. To explore the free energy landscapes of proteins over large conformational space, the stability of native structure is perturbed by biochemical or mechanical means, and the conformational transition process is measured. In single molecular manipulation experiments, stretching force is applied to proteins, and the folding and unfolding transitions are recorded by the extension time course. Due to the broad force range and long-time stability of magnetic tweezers, the free energy landscape over large conformational space can be obtained. In this article, we describe the magnetic tweezers instrument design, protein construct design and preparation, fluid chamber preparation, common-used measuring protocols including force-ramp and force-jump measurements, and data analysis methods to construct the free energy landscape. Single-domain cold shock protein is introduced as an example to build its free energy landscape by magnetic tweezers measurements.

Robust magnetic tweezers for membrane protein folding studies.

Single-molecule magnetic tweezers have recently been adapted for monitoring the interactions between transmembrane helices of membrane proteins within lipid bilayers. In this chapter, we describe the procedures of conducting studies on membrane protein folding using a robust magnetic tweezer method. This tweezer method is capable of observing thousands of (un)folding transitions over extended periods of several to tens of hours. Using this approach, we can dissect the folding pathways of membrane proteins, determine their folding time scales, and map the folding energy landscapes, with a higher statistical reliability. Our robust magnetic tweezers also allow for estimating the folding speed limit of helical membrane proteins, which serves as a link between the kinetics and barrier energies.

Force-fluorescence setup for observing protein-DNA interactions under load.

This chapter explores advanced single-molecule techniques for studying protein-DNA interactions, particularly focusing on Replication Protein A (RPA) using a force-fluorescence setup. It combines magnetic tweezers (MT) with total internal reflection fluorescence (TIRF) microscopy, enabling detailed observation of DNA behavior under mechanical stress. The chapter details the use of DNA hairpins and bare DNA to examine RPA's binding dynamics and its influence on DNA's mechanical properties. This approach provides deeper insights into RPA's role in DNA replication, repair, and recombination, highlighting its significance in maintaining genomic stability.

Magnetic tweezers characterization of the entropic elasticity of intrinsically disordered proteins and peptoids.

Understanding the conformational behavior of biopolymers is essential to unlocking knowledge of their biophysical mechanisms and functional roles. Single-molecule force spectroscopy can provide a unique perspective on this by exploiting entropic elasticity to uncover key biopolymer structural parameters. A particularly powerful approach involves the use of magnetic tweezers, which can easily generate lower stretching forces (0.1-20 pN). For forces at the low end of this range, the elastic response of biopolymers is sensitive to excluded volume effects, and they can be described by Pincus blob elasticity model that allow robust extraction of the Flory polymer scaling exponent. Here, we detail protocols for the use of magnetic tweezers for force-extension measurements of intrinsically disordered proteins and peptoids. We also discuss procedures for fitting low-force elastic curves to the predictions of polymer physics models to extract key conformational parameters.

Use of DNA forceps to measure receptor-ligand dissociation equilibrium constants in a single-molecule competition assay.

The ability of biophysicists to decipher the behavior of individual biomolecules has steadily improved over the past thirty years. However, it still remains unclear how an ensemble of data acquired at the single-molecule level compares with the data acquired on an ensemble of the same molecules. We here propose an assay to tackle this question in the context of dissociation equilibrium constant measurements. A sensor is built by engrafting a receptor and a ligand onto a flexible dsDNA scaffold and mounting this assembly on magnetic tweezers. This way, looking at the position of the magnetic bead enables one to determine in real-time if the two molecular partners are associated or not. Next, to quantify the affinity of the scrutinized single-receptor for a given competitor, various amounts of the latter molecule are introduced in solution and the equilibrium response of the sensor is monitored throughout the titration protocol. Proofs of concept are established for the binding of three rapamycin analogs to the FKBP12 cis-trans prolyl isomerase. For each of these drugs the mean affinity constant obtained on a ten of individual receptors agrees with the one previously determined in a bulk assay. Furthermore, experimental contingencies are sufficient to explain the dispersion observed over the single-molecule values.

Construction and operation of high-resolution magnetic tape head tweezers for measuring single-protein dynamics under force.

Mechanical forces are critical to protein function across many biological contexts-from bacterial adhesion to muscle mechanics and mechanotransduction processes. Hence, understanding how mechanical forces govern protein activity has developed into a central scientific question. In this context, single-molecule magnetic tweezers has recently emerged as a valuable experimental tool, offering the capability to measure single proteins over physiologically relevant forces and timescales. In this chapter, we present a detailed protocol for the assembly and operation of our magnetic tape head tweezers instrument, specifically tailored to investigate protein dynamics. Our instrument boasts a simplified microscope design and incorporates a magnetic tape head as the force-generating apparatus, facilitating precise force control and enhancing its temporal stability, enabling the study of single protein mechanics over extended timescales spanning several hours or even days. Moreover, its straightforward and cost-effective design ensures its accessibility to the wider scientific community. We anticipate that this technique will attract widespread interest within the growing field of mechanobiology and expect that this chapter will provide facilitated accessibility to this technology.

Single-molecule tethering methods for membrane proteins.

Molecular tethering of a single membrane protein between the glass surface and a magnetic bead is essential for studying the structural dynamics of membrane proteins using magnetic tweezers. However, the force-induced bond breakage of the widely-used digoxigenin-antidigoxigenin tether complex has imposed limitations on its stable observation. In this chapter, we describe the procedures of constructing highly stable single-molecule tethering methods for membrane proteins. These methods are established using dibenzocyclooctyne click chemistry, traptavidin-biotin binding, SpyCatcher-SpyTag conjugation, and SnoopCatcher-SnoopTag conjugation. The molecular tethering approaches allow for more stable observation of structural transitions in membrane proteins under force.

Manipulation of Embryonic Cleavage Geometry Using Magnetic Tweezers.

The geometry of reductive divisions that mark the development of early embryos instructs cell fates, sizes, and positions, by mechanisms that remain unclear. In that context, new methods to mechanically manipulate these divisions are starting to emerge in different model systems. These are key to develop future innovative approaches and understand developmental mechanisms controlled by cleavage geometry. In particular, how cell cycle pace is regulated in rapidly reducing blastomeres and how fate diversity can arise from blastomere size and position within embryos are fundamental questions that remain at the heart of ongoing research. In this chapter, we provide a detailed protocol to assemble and use magnetic tweezers in the sea urchin model and generate spatially controlled asymmetric and oriented divisions during early embryonic development.

Magnetically navigated microbubbles coated with albumin/polyarginine and superparamagnetic iron oxide nanoparticles.

While microbubbles (MB) are routinely used for ultrasound (US) imaging, magnetic MB are increasingly explored as they can be guided to specific sites of interest by applied magnetic field gradient. This requires the MB shell composition tuning to prolong MB stability and provide functionalization capabilities with magnetic nanoparticles. Hence, we developed air-filled MB stabilized by a protein-polymer complex of bovine serum albumin (BSA) and poly-L-arginine (pArg) of different molecular weights, showing that pArg of moderate molecular weight distribution (15-70 kDa) enabled MB with greater stability and acoustic response while preserving MB narrow diameters and the relative viability of THP-1 cells after 48 h of incubation. After MB functionalization with superparamagnetic iron oxide nanoparticles (SPION), magnetic moment values provided by single MB confirmed the sufficient SPION deposition onto BSA + pArg MB shells. During MB magnetic navigation in a blood vessel mimicking phantom with magnetic tweezers and in a Petri dish with adherent mouse renal carcinoma cell line, we demonstrated the effectiveness of magnetic MB localization in the desired area by magnetic field gradient. Magnetic MB co-localization with cells was further exploited for effective doxorubicin delivery with drug-loaded MB. Taken together, these findings open new avenues in control over albumin MB properties and magnetic navigation of SPION-loaded MB, which can envisage their applications in diagnostic and therapeutic needs.

Elucidating the novel mechanisms of molecular chaperones by single-molecule technologies.

Molecular chaperones play central roles in sustaining protein homeostasis and preventing protein aggregation. Most studies of these systems have been performed in bulk, providing averaged measurements, though recent single-molecule approaches have provided an in-depth understanding of the molecular mechanisms of their activities and structural rearrangements during substrate recognition. Chaperone activities have been observed to be substrate specific, with some associated with ATP-dependent structural dynamics and others via interactions with co-chaperones. This Review aims to describe the novel mechanisms of molecular chaperones as revealed by single-molecule approaches, and to provide insights into their functioning and its implications for protein homeostasis and human diseases.

Connecting the dots: key insights on ParB for chromosome segregation from single-molecule studies.

Bacterial cells require DNA segregation machinery to properly distribute a genome to both daughter cells upon division. The most common system involved in chromosome and plasmid segregation in bacteria is the ParABS system. A core protein of this system - partition protein B (ParB) - regulates chromosome organization and chromosome segregation during the bacterial cell cycle. Over the past decades, research has greatly advanced our knowledge of the ParABS system. However, many intricate details of the mechanism of ParB proteins were only recently uncovered using in vitro single-molecule techniques. These approaches allowed the exploration of ParB proteins in precisely controlled environments, free from the complexities of the cellular milieu. This review covers the early developments of this field but emphasizes recent advances in our knowledge of the mechanistic understanding of ParB proteins as revealed by in vitro single-molecule methods. Furthermore, we provide an outlook on future endeavors in investigating ParB, ParB-like proteins, and their interaction partners.

Piconewton Forces Mediate GAIN Domain Dissociation of the Latrophilin-3 Adhesion GPCR.

Latrophilins are adhesion G-protein coupled receptors (aGPCRs) that control excitatory synapse formation. Most aGPCRs, including latrophilins, are autoproteolytically cleaved at their GPCR-autoproteolysis inducing (GAIN) domain, but the two resulting fragments remain noncovalently associated on the cell surface. Force-mediated dissociation of the fragments is thought to activate G-protein signaling, but how this mechanosensitivity arises is poorly understood. Here, we use magnetic tweezer assays to show that physiologically relevant forces in the 1-10 pN range lead to dissociation of the latrophilin-3 GAIN domain on the seconds-to-minutes time scale, compared to days in the absence of force. In addition, we find that the GAIN domain undergoes large changes in length in response to increasing mechanical load. These data are consistent with a model in which a force-sensitive equilibrium between compact and extended GAIN domain states precedes dissociation, suggesting a mechanism by which latrophilins and other aGPCRs may mediate mechanically induced signal transduction.