Single-molecule Studies Research Articles

A single-molecule conductance study on the rotational isomers of a hexaarylbenzene derivative carrying dipolar rotating units

Using a scanning tunneling microscopy-break junction (STM-BJ) technique, we evaluated the single-molecule conductance of stable but interconvertible two rotational isomers of a hexaarylbenzene derivative featuring dipolar rotatable difluorophenyl units and tripodal thiol groups. The STM-BJ measurements of a configuration of single-molecule junction, formed between a Au-STM tip and a Au(111)/mica substrate covered with the hexaarylbenzene molecule, showed that the conductance behaviors of the rotational isomers are different from one another. This result takes a step forward in our goal of developing single-molecule switching/memory systems driven by electric fields.

Molecular Mechanism of Processive Stepping of Kinesin Motors

Kinesin-1 is a motor protein that can step processively on microtubule by hydrolyzing ATP molecules, playing an essential role in intracellular transports. To better understand the mechanochemical coupling of the motor stepping cycle, numerous structural, biochemical, single molecule, theoretical modeling and numerical simulation studies have been undertaken for the kinesin-1 motor. Recently, a novel ultraresolution optical trapping method was employed to study the mechanics of the kinesin-1 motor and new results were supplemented to its stepping dynamics. In this commentary, the new single molecule results are explained well theoretically with one of the models presented in the literature for the mechanochemical coupling of the kinesin-1 motor. With the model, various prior experimental results for dynamics of different families of N-terminal kinesin motors have also been explained quantitatively.

Single-molecule tracking technologies for quantifying the dynamics of gene regulation in cells, tissue and embryos.

For decades, we have relied on population and time-averaged snapshots of dynamic molecular scale events to understand how genes are regulated during development and beyond. The advent of techniques to observe single-molecule kinetics in increasingly endogenous contexts, progressing from in vitro studies to living embryos, has revealed how much we have missed. Here, we provide an accessible overview of the rapidly expanding family of technologies for single-molecule tracking (SMT), with the goal of enabling the reader to critically analyse single-molecule studies, as well as to inspire the application of SMT to their own work. We start by overviewing the basics of and motivation for SMT experiments, and the trade-offs involved when optimizing parameters. We then cover key technologies, including fluorescent labelling, excitation and detection optics, localization and tracking algorithms, and data analysis. Finally, we provide a summary of selected recent applications of SMT to study the dynamics of gene regulation.

DNA Origami Nanoantennas for Fluorescence Enhancement.

The possibility to increase fluorescence by plasmonic effects in the near-field of metal nanostructures was recognized more than half a century ago. A major challenge, however, was to use this effect because placing single quantum emitters in the nanoscale plasmonic hotspot remained unsolved for a long time. This not only presents a chemical problem but also requires the nanostructure itself to be coaligned with the polarization of the excitation light. Additional difficulties arise from the complex distance dependence of fluorescence emission: in contrast to other surface-enhanced spectroscopies (such as Raman spectroscopy), the emitter should not be placed as close as possible to the metallic nanostructure but rather needs to be at an optimal distance on the order of a few nanometers to avoid undesired quenching effects.Our group addressed these challenges almost a decade ago by exploiting the unique positioning ability of DNA nanotechnology and reported the first self-assembled DNA origami nanoantennas. This Account summarizes our work spanning from this first proof-of-principle study to recent advances in utilizing DNA origami nanoantennas for single DNA molecule detection on a portable smartphone microscope.We summarize different aspects of DNA origami nanoantennas that are essential for achieving strong fluorescence enhancement and discuss how single-molecule fluorescence studies helped us to gain a better understanding of the interplay between fluorophores and plasmonic hotspots. Practical aspects of preparing the DNA origami nanoantennas and extending their utility are also discussed.Fluorescence enhancement in DNA origami nanoantennas is especially exciting for signal amplification in molecular diagnostic assays or in single-molecule biophysics, which could strongly benefit from higher time resolution. Additionally, biophysics can greatly profit from the ultrasmall effective detection volumes provided by DNA nanoantennas that allow single-molecule detection at drastically elevated concentrations as is required, e.g., in single-molecule DNA sequencing approaches.Finally, we describe our most recent progress in developing DNA NanoAntennas with Cleared HOtSpots (NACHOS) that are fully compatible with biomolecular assays. The developed DNA origami nanoantennas have proven robustness and remain functional after months of storage. As an example, we demonstrated for the first time the single-molecule detection of DNA specific to antibiotic-resistant bacteria on a portable and battery-driven smartphone microscope enabled by DNA origami nanoantennas. These recent developments mark a perfect moment to summarize the principles and the synthesis of DNA origami nanoantennas and give an outlook of new exciting directions toward using different nanomaterials for the construction of nanoantennas as well as for their emerging applications.

Self-Induced Heterogeneity in Deeply Supercooled Liquids.

A theoretical treatment of deeply supercooled liquids is difficult because their properties emerge from spatial inhomogeneities that are self-induced, transient, and nanoscopic. I use computer simulations to analyze self-induced static and dynamic heterogeneity in equilibrium systems approaching the experimental glass transition. I characterize the broad sample-to-sample fluctuations of salient dynamic and thermodynamic properties in elementary mesoscopic systems. Findings regarding local lifetimes and distributions of dynamic heterogeneity are in excellent agreement with recent single molecule studies. Surprisingly broad thermodynamic fluctuations are also found, which correlate well with dynamic fluctuations, thus providing a local test of the thermodynamic origin of slow dynamics.

Single-molecule studies reveal branched pathways for activator-dependent assembly of RNA polymerase II pre-initiation complexes

RNA polymerase II (RNA Pol II) transcription reconstituted from purified factors suggests pre-initiation complexes (PICs) can assemble by sequential incorporation of factors at the TATA box. However, these basal transcription reactions are generally independent of activators and co-activators. To study PIC assembly under more realistic conditions, we used single-molecule microscopy to visualize factor dynamics during activator-dependent reactions in nuclear extracts. Surprisingly, RNA Pol II, TFIIF, and TFIIE can pre-assemble on enhancer-bound activators before loading into PICs, and multiple RNA Pol II complexes can bind simultaneously to create a localized cluster. Unlike TFIIF and TFIIE, TFIIH binding is singular and dependent on the basal promoter. Activator-tethered factors exhibit dwell times on the order of seconds. In contrast, PICs can persist on the order of minutes in the absence of nucleotide triphosphates, although TFIIE remains unexpectedly dynamic even after TFIIH incorporation. Our kinetic measurements lead to a new branched model for activator-dependent PIC assembly.

Stochastic Noise in Single-Nanoparticle Catalysis

Nanoparticle-based catalysts exhibit considerable complexity and heterogeneity. New light can be shed into these aspects by probing catalysis on the level of single nanoparticles. Here, we monitor on individual Ag nanoparticles plasmon-excitation-driven CO2 reduction and formation of multicarbon species on the Ag surface. The measurements show that catalytic activities and selectivities are variable from one nanoparticle to another, an effect that would typically be attributed to structural and morphological differences between nanoparticles. However, the nanoparticle-to-nanoparticle variations observed here are accompanied by temporal fluctuations in the catalytic activity. We infer that the variation captures, in addition to static heterogeneities in the nanoparticle-based catalyst, noise from inherent stochasticity of catalytic events occurring on the nanoparticle surface. Such noise can be an inadvertent source of variations observed in single-nanoparticle, single-molecule studies of catalysis.

A Horizontal Magnetic Tweezers for Studying Single DNA Molecules and DNA-Binding Proteins.

We report data from single molecule studies on the interaction between single DNA molecules and core histones using custom-designed horizontal magnetic tweezers. The DNA-core histone complexes were formed using λ-DNA tethers, core histones, and NAP1 and were exposed to forces ranging from ~2 pN to ~74 pN. During the assembly events, we observed the length of the DNA decrease in approximate integer multiples of ~50 nm, suggesting the binding of the histone octamers to the DNA tether. During the mechanically induced disassembly events, we observed disruption lengths in approximate integer multiples of ~50 nm, suggesting the unbinding of one or more octamers from the DNA tether. We also observed histone octamer unbinding events at forces as low as ~2 pN. Our horizontal magnetic tweezers yielded high-resolution, low-noise data on force-mediated DNA-core histone assembly and disassembly processes.

Elucidating the Mechanism Behind Sodium-Coupled Neurotransmitter Transporters by Reconstitution.

Sodium-coupled neurotransmitter transporters play a fundamental role in the termination of synaptic neurotransmission, which makes them a major drug target. The reconstitution of these secondary active transporters into liposomes has shed light on their molecular transport mechanisms. From the earliest days of the reconstitution technique up to today's single-molecule studies, insights from live functioning transporters have been indispensable for our understanding of their physiological impact. The two classes of sodium-coupled neurotransmitter transporters, the neurotransmitter: sodium symporters and the excitatory amino acid transporters, have vastly different molecular structures, but complementary proteoliposome studies have sought to unravel their ion-dependence and transport kinetics. Furthermore, reconstitution experiments have been used on both protein classes to investigate the role of e.g. the lipid environment, of posttranslational modifications, and of specific amino acid residues in transport. Techniques that allow the detection of transport at a single-vesicle resolution have been developed, and single-molecule studies have started to reveal single transporter kinetics, which will expand our understanding of how transport across the membrane is facilitated at protein level. Here, we review a selection of the results and applications where the reconstitution of the two classes of neurotransmitter transporters has been instrumental.

Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility.

The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule-binding domains (MTBDs) that step along the track. Here, we simultaneously tracked with nanometer precision two MTBDs and one AAA ring of a single dynein as it underwent hundreds of steps using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take differently sized steps. This variability in the movement between the AAA ring and MTBDs results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.

Optical microlever assisted DNA stretching.

Optical microrobotics is an emerging field that has the potential to improve upon current optical tweezer studies through avenues such as limiting the exposure of biological molecules of interest to laser radiation and overcoming the current limitations of low forces and unwanted interactions between nearby optical traps. However, optical microrobotics has been historically limited to rigid, single-body end-effectors rather than even simple machines, limiting the tasks that can be performed. Additionally, while multi-body machines such as microlevers exist in the literature, they have not yet been successfully demonstrated as tools for biological studies, such as molecule stretching. In this work we have taken a step towards moving the field forward by developing two types of microlever, produced using two-photon absorption polymerisation, to perform the first lever-assisted stretches of double-stranded DNA. The aim of the work is to provide a proof of concept for using optical micromachines for single molecule studies. Both styles of microlevers were successfully used to stretch single duplexes of DNA, and the results were analysed with the worm-like chain model to show that they were in good agreement.

A Versatile Sub-Nanomolar Fluorescent Ligand Enables NanoBRET Binding Studies and Single-Molecule Microscopy at the Histamine H3 Receptor.

The histamine H3 receptor (H3R) is considered an attractive drug target for various neurological diseases. We here report the synthesis of UR-NR266, a novel fluorescent H3R ligand. Broad pharmacological characterization revealed UR-NR266 as a sub-nanomolar compound at the H3R with an exceptional selectivity profile within the histamine receptor family. The presented neutral antagonist showed fast association to its target and complete dissociation in kinetic binding studies. Detailed characterization of standard H3R ligands in NanoBRET competition binding using UR-NR266 highlights its value as a versatile pharmacological tool to analyze future H3R ligands. The low nonspecific binding observed in all experiments could also be verified in TIRF and confocal microscopy. This fluorescent probe allows the highly specific analysis of native H3R in various assays ranging from optical high throughput technologies to biophysical analyses and single-molecule studies in its natural environment. An off-target screening at 14 receptors revealed UR-NR266 as a selective compound.

Potential of Single-Molecule Live-Cell Imaging for Chemical Translational Biology.

Single-molecule live-cell imaging is the most direct approach for monitoring the motility of molecules in living cells. Considering the relationship between the motility of molecules and their function, information obtained from single-molecule imaging involves essential clues for understanding the regulatory mechanisms of the processes of target molecules, and translation to applied sciences such as drug discovery. In this Concept, examples of single-molecule imaging studies on G protein-coupled receptors (GPCRs) are mainly introduced, and recent techniques of single-molecule imaging for overcoming the limitation of single-molecule live-cell imaging are discussed. Based on these studies, the prospects of single-molecule imaging will be outlined.

Molecular insight into how γ-TuRC makes microtubules.

As one of four filament types, microtubules are a core component of the cytoskeleton and are essential for cell function. Yet how microtubules are nucleated from their building blocks, the αβ-tubulin heterodimer, has remained a fundamental open question since the discovery of tubulin 50 years ago. Recent structural studies have shed light on how γ-tubulin and the γ-tubulin complex proteins (GCPs) GCP2 to GCP6 form the γ-tubulin ring complex (γ-TuRC). In parallel, functional and single-molecule studies have informed on how the γ-TuRC nucleates microtubules in real time, how this process is regulated in the cell and how it compares to other modes of nucleation. Another recent surprise has been the identification of a second essential nucleation factor, which turns out to be the well-characterized microtubule polymerase XMAP215 (also known as CKAP5, a homolog of chTOG, Stu2 and Alp14). This discovery helps to explain why the observed nucleation activity of the γ-TuRC in vitro is relatively low. Taken together, research in recent years has afforded important insight into how microtubules are made in the cell and provides a basis for an exciting era in the cytoskeleton field.

Atomic force microscopy-A tool for structural and translational DNA research.

Atomic force microscopy (AFM) is a powerful imaging technique that allows for structural characterization of single biomolecules with nanoscale resolution. AFM has a unique capability to image biological molecules in their native states under physiological conditions without the need for labeling or averaging. DNA has been extensively imaged with AFM from early single-molecule studies of conformational diversity in plasmids, to recent examinations of intramolecular variation between groove depths within an individual DNA molecule. The ability to image dynamic biological interactions in situ has also allowed for the interaction of various proteins and therapeutic ligands with DNA to be evaluated—providing insights into structural assembly, flexibility, and movement. This review provides an overview of how innovation and optimization in AFM imaging have advanced our understanding of DNA structure, mechanics, and interactions. These include studies of the secondary and tertiary structure of DNA, including how these are affected by its interactions with proteins. The broader role of AFM as a tool in translational cancer research is also explored through its use in imaging DNA with key chemotherapeutic ligands, including those currently employed in clinical practice.

Single-Molecule Study of Thermomyces lanuginosus Lipase in a Detergency Application System Reveals Diffusion Pattern Remodeling by Surfactants and Calcium.

Lipases comprise one of the major enzyme classes in biotechnology with applications within, e.g., baking, brewing, biocatalysis, and the detergent industry. Understanding the mechanisms of lipase function and regulation is therefore important to facilitate the optimization of their function by protein engineering. Advances in single-molecule studies in model systems have provided deep mechanistic insights on lipase function, such as the existence of functional states, their dependence on regulatory cues, and their correlation to activity. However, it is unclear how these observations translate to enzyme behavior in applied settings. Here, single-molecule tracking of individual Thermomyces lanuginosus lipase (TLL) enzymes in a detergency application system allowed real-time direct observation of spatiotemporal localization, and thus diffusional behavior, of TLL enzymes on a lard substrate. Parallelized imaging of thousands of individual enzymes allowed us to observe directly the existence and quantify the abundance and interconversion kinetics between three diffusional states that we recently provided evidence to correlate with function. We observe redistribution of the enzyme's diffusional pattern at the lipid-water interface as well as variations in binding efficiency in response to surfactants and calcium, demonstrating that detergency effectors can drive the sampling of lipase functional states. Our single-molecule results combined with ensemble activity assays and enzyme surface binding efficiency readouts allowed us to deconvolute how application conditions can significantly alter protein functional dynamics and/or surface binding, both of which underpin enzyme performance. We anticipate that our results will inspire further efforts to decipher and integrate the dynamic nature of lipases, and other enzymes, in the design of new biotechnological solutions.

Single molecule studies reveal branched pathways for activator-dependent assembly of RNA polymerase II preinitiation complexes

Single molecule studies reveal branched pathways for activator-dependent assembly of RNA polymerase II preinitiation complexes

Optical manipulation: advances for biophotonics in the 21st century.

.Significance: Optical trapping is a technique capable of applying minute forces that has been applied to studies spanning single molecules up to microorganisms.Aim: The goal of this perspective is to highlight some of the main advances in the last decade in this field that are pertinent for a biomedical audience.Approach: First, the direct determination of forces in optical tweezers and the combination of optical and acoustic traps, which allows studies across different length scales, are discussed. Then, a review of the progress made in the direct trapping of both single-molecules, and even single-viruses, and single cells with optical forces is outlined. Lastly, future directions for this methodology in biophotonics are discussed.Results: In the 21st century, optical manipulation has expanded its unique capabilities, enabling not only a more detailed study of single molecules and single cells but also of more complex living systems, giving us further insights into important biological activities.Conclusions: Optical forces have played a large role in the biomedical landscape leading to exceptional new biological breakthroughs. The continuous advances in the world of optical trapping will certainly lead to further exploitation, including exciting in-vivo experiments.

Biophysical properties of the isolated spike protein binding helix of human ACE2

The entry of the severe acute respiratory syndrome coronavirus 2 virus in human cells is mediated by the binding of its surface spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor. A 23-residue long helical segment (SBP1) at the binding interface of human ACE2 interacts with viral spike protein and therefore has generated considerable interest as a recognition element for virus detection. Unfortunately, emerging reports indicate that the affinity of SBP1 to the receptor-binding domain of the spike protein is much lower than that of the ACE2 receptor itself. Here, we examine the biophysical properties of SBP1 to reveal factors leading to its low affinity for the spike protein. Whereas SBP1 shows good solubility (solubility > 0.8 mM), circular dichroism spectroscopy shows that it is mostly disordered with some antiparallel β-sheet content and no helicity. The helicity is substantial (>20%) only upon adding high concentrations (≥20% v/v) of 2,2,2-trifluoroethanol, a helix promoter. Fluorescence correlation spectroscopy and single-molecule photobleaching studies show that the peptide oligomerizes at concentrations >50 nM. We hypothesized that mutating the hydrophobic residues (F28, F32, and F40) of SBP1, which do not directly interact with the spike protein, to alanine would reduce peptide oligomerization without affecting its spike binding affinity. Whereas the mutant peptide (SBP1mod) shows substantially reduced oligomerization propensity, it does not show improved helicity. Our study shows that the failure of efforts, so far, to produce a short SBP1 mimic with a high affinity for the spike protein is not only due to the lack of helicity but is also due to the heretofore unrecognized problem of oligomerization.

Molecular design of the γδT cell receptor ectodomain encodes biologically fit ligand recognition in the absence of mechanosensing

High-acuity αβT cell receptor (TCR) recognition of peptides bound to major histocompatibility complex molecules (pMHCs) requires mechanosensing, a process whereby piconewton (pN) bioforces exert physical load on αβTCR-pMHC bonds to dynamically alter their lifetimes and foster digital sensitivity cellular signaling. While mechanotransduction is operative for both αβTCRs and pre-TCRs within the αβT lineage, its role in γδT cells is unknown. Here, we show that the human DP10.7 γδTCR specific for the sulfoglycolipid sulfatide bound to CD1d only sustains a significant load and undergoes force-induced structural transitions when the binding interface-distal γδ constant domain (C) module is replaced with that of αβ. The chimeric γδ-αβTCR also signals more robustly than does the wild-type (WT) γδTCR, as revealed by RNA-sequencing (RNA-seq) analysis of TCR-transduced Rag2-/- thymocytes, consistent with structural, single-molecule, and molecular dynamics studies reflective of γδTCRs as mediating recognition via a more canonical immunoglobulin-like receptor interaction. Absence of robust, force-related catch bonds, as well as γδTCR structural transitions, implies that γδT cells do not use mechanosensing for ligand recognition. This distinction is consonant with the fact that their innate-type ligands, including markers of cellular stress, are expressed at a high copy number relative to the sparse pMHC ligands of αβT cells arrayed on activating target cells. We posit that mechanosensing emerged over ∼200 million years of vertebrate evolution to fulfill indispensable adaptive immune recognition requirements for pMHC in the αβT cell lineage that are unnecessary for the γδT cell lineage mechanism of non-pMHC ligand detection.