Optical tweezers have become the method of choice in single-molecule manipulation studies. In this Primer, we first review the physical principles of optical tweezers and the characteristics that make them a powerful tool to investigate single molecules. We then introduce the modifications of the method to extend the measurement of forces and displacements to torques and angles, and to develop optical tweezers with single-molecule fluorescence detection capabilities. We discuss force and torque calibration of these instruments, their various modes of operation and most common experimental geometries. We describe the type of data obtained in each experimental design and their analyses. This description is followed by a survey of applications of these methods to the studies of protein–nucleic acid interactions, protein/RNA folding and molecular motors. We also discuss data reproducibility, the factors that lead to the data variability among different laboratories and the need to develop field standards. We cover the current limitations of the methods and possible ways to optimize instrument operation, data extraction and analysis, before suggesting likely areas of future growth. This Primer on optical tweezers describes the instrumentation and experimental designs used in most single-molecule optical tweezers assays and discusses optical tweezers measurements in systems of biophysical interest such as DNA elasticity, protein and RNA folding, and molecular motors.

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Optical tweezers in single-molecule biophysics.

The relationship between protein three-dimensional structure and function is essential for mechanism determination. Unfortunately, most techniques do not provide a direct measurement of this relationship. Structural data are typically limited to static pictures, and function must be inferred. Conversely, functional assays usually provide little information on structural conformation. We developed a single-molecule technique combining optical tweezers and fluorescence microscopy that allows for both measurements simultaneously. Here we present measurements of UvrD, a DNA repair helicase, that directly and unambiguously reveal the connection between its structure and function. Our data reveal that UvrD exhibits two distinct types of unwinding activity regulated by its stoichiometry. Furthermore, two UvrD conformational states, termed “closed” and “open,” correlate with movement toward or away from the DNA fork.

https://science.sciencemag.org/content/sci/348/6232/352.full.pdf

Direct observation of structure-function relationship in a nucleic acid–processing enzyme

https://epublications.marquette.edu/cgi/viewcontent.cgi?article=1601&context=bio_fac

Dynamics of E. coli single stranded DNA binding (SSB) protein-DNA complexes

Notch signaling, involved in development and tissue homeostasis, is activated at the cell–cell interface through ligand–receptor interactions. Previous studies have implicated mechanical forces in the activation of Notch receptor upon binding to its ligand. Here we aimed to determine the single molecular force required for Notch activation by developing a novel low tension gauge tether (LTGT). LTGT utilizes the low unbinding force between single-stranded DNA (ssDNA) and Escherichia coli ssDNA binding protein (SSB) (∼4 pN dissociation force at 500 nm/s pulling rate). The ssDNA wraps around SSB and, upon application of force, unspools from SSB, much like the unspooling of a yoyo. One end of this nano yoyo is attached to the surface though SSB, while the other end presents a ligand. A Notch receptor, upon binding to its ligand, is believed to undergo force-induced conformational changes required for activating downstream signaling. If the required force for such activation is larger than 4 pN, ssDNA will uns...

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Defining Single Molecular Forces Required for Notch Activation Using Nano Yoyo

Over the past two decades, single-molecule techniques have evolved into robust tools to study many fundamental biological processes. The combination of optical tweezers with fluorescence microscopy and microfluidics provides a powerful single-molecule manipulation and visualization technique that has found widespread application in biology. In this combined approach, the spatial (~nm) and temporal (~ms) resolution, as well as the force scale (~pN) accessible to optical tweezers is complemented with the power of fluorescence microscopy. Thereby, it provides information on the local presence, identity, spatial dynamics, and conformational dynamics of single biomolecules. Together, these techniques allow comprehensive studies of, among others, molecular motors, protein-protein and protein-DNA interactions, biomolecular conformational changes, and mechanotransduction pathways. In this chapter, recent applications of fluorescence microscopy in combination with optical trapping are discussed. After an introductory section, we provide a description of instrumentation together with the current capabilities and limitations of the approaches. Next we summarize recent studies that applied this combination of techniques in biological systems and highlight some representative biological assays to mark the exquisite opportunities that optical tweezers combined with fluorescence microscopy provide.

Recent Advances in Biological Single-Molecule Applications of Optical Tweezers and Fluorescence Microscopy

Conformational control of biomolecular activities can reveal functional insights and enable the engineering of novel activities. Here we show that conformational control through intramolecular cross-linking of a helicase monomer with undetectable unwinding activity converts it into a superhelicase that can unwind thousands of base pairs processively, even against a large opposing force. A natural partner that enhances the helicase activity is shown to achieve its stimulating role also by selectively stabilizing the active conformation. Our work provides insight into the regulation of nucleic acid unwinding activity and introduces a monomeric superhelicase without nuclease activities, which may be useful for biotechnological applications.

https://science.sciencemag.org/content/sci/348/6232/344.full.pdf

Engineering of a superhelicase through conformational control

Escherichia coli single-stranded (ss)DNA binding (SSB) protein mediates genome maintenance processes by regulating access to ssDNA. This homotetrameric protein wraps ssDNA in multiple distinct binding modes that may be used selectively in different DNA processes, and whose detailed wrapping topologies remain speculative. Here, we used single-molecule force and fluorescence spectroscopy to investigate E. coli SSB binding to ssDNA. Stretching a single ssDNA-SSB complex reveals discrete states that correlate with known binding modes, the likely ssDNA conformations and diffusion dynamics in each, and the kinetic pathways by which the protein wraps ssDNA and is dissociated. The data allow us to construct an energy landscape for the ssDNA-SSB complex, revealing that unwrapping energy costs increase the more ssDNA is unraveled. Our findings provide insights into the mechanism by which proteins gain access to ssDNA bound by SSB, as demonstrated by experiments in which SSB is displaced by the E. coli recombinase RecA.

/pdf/structural-dynamics-of-e-coli-single-stranded-dna-binding-14oihceels.pdf

Structural dynamics of E. coli single-stranded DNA binding protein reveal DNA wrapping and unwrapping pathways

High-plex imaging of RNA and proteins at subcellular resolution in fixed tissue by spatial molecular imaging.


 Cancer research across drug development, molecular biomarkers, and patient response depends on understanding biology that is dependent on complex interactions between malignant, immune, and stromal cells. To survive clearance mechanisms, a tumor can rely on a myriad of escape strategies, and the microenvironment is architected around the current path of escape. To enable a more comprehensive picture of tumor biology, we have developed the CosMx™ Spatial Molecular Imager (SMI) technology to capture a snapshot of thousands of RNA species resolved subcellularly from a single, standard histopathology slide. Building upon the previously released panels, this study tests a new 6,000-plex panel, the highest RNA plex measured in situ within human tissue, allowing the imputation of a spatial whole transcriptome in the tissue. We performed an ultra-high-plex RNA assay to detect 6,000 targets simultaneously in situ on an FFPE human liver cancer tissue (~1 cm2 area) using the CosMx SMI. This RNA panel covers broad biological areas with special emphasis on oncology, immunology, and signal transduction, such that all cancer researchers can benefit from the direct detection of targets of interest (sans imputation) in intact tissue. Analysis algorithms were developed to allow robust assessments of cell types, cell states, cell-cell interactions, and pathway activation. Imputation based on reference profiles from HCA, TCGA, and other public repositories allows estimation of non-measured transcripts at a ratio of approximately 1:3, compared to the approximate 1:20-1:70 imputations performed previously for spatial data.Thousands of transcripts were simultaneously detected with high sensitivity and specificity on the FFPE liver cancer tissue section at single-cell subcellular resolution. We were able to accurately map known reference profiles from scRNA-seq into this sample while identifying cancer-specific malignant, immune, and stromal cells in this tissue sample using this ultra-high plex RNA panel. In addition, we constructed sample-specific spatial neighborhoods, defined by cell types, cell states, and nearly unlimited sets of biological pathways through the imputed whole transcriptome. Finally, we measured >1,000 ligand-receptor interactions between key cell types of adjacent cells in the tissue, identifying mechanisms for tumor-mediated escape as well as reactive re-architecting of the native stroma which defines the trajectory of cancer’s evolution. Single-cell spatial measurements of gene expression at 6,000 plex from a single FFPE slide has the potential to transform our understanding of tumor biology and facilitate the next advances in cancer research by extracting the highest data density possible from rare specimens collected during patient treatment.
 Citation Format: Shanshan He, Michael Patrick, Jason W. Reeves, Patrick Danaher, Julian Preciado, Joseph Phan, Erin Piazza, Zachary Reitz, Lidan Wu, Rustem Khafizov, Haiyan Zhai, Michael Rhodes, David Ruff, Joseph Beechem. Path to the holy grail of spatial biology: Spatial single-cell whole transcriptomes using 6000-plex spatial molecular imaging on FFPE tissue. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5637.

Abstract 5637: Path to the holy grail of spatial biology: Spatial single-cell whole transcriptomes using 6000-plex spatial molecular imaging on FFPE tissue

Rustem Khafizov

Papers

Engineering of a superhelicase through conformational control

Structural dynamics of E. coli single-stranded DNA binding protein reveal DNA wrapping and unwrapping pathways

High-plex imaging of RNA and proteins at subcellular resolution in fixed tissue by spatial molecular imaging.

Abstract 5637: Path to the holy grail of spatial biology: Spatial single-cell whole transcriptomes using 6000-plex spatial molecular imaging on FFPE tissue