Special Issue "Single-Molecule Techniques"

A special issue of Methods and Protocols (ISSN 2409-9279).

Deadline for manuscript submissions: closed (31 October 2018)

Special Issue Editor

Guest Editor
Dr. Soma Dhakal

Virginia Commonwealth University, Richmond, VA, USA
Website | E-Mail
Interests: single-molecule analysis of protein-DNA interaction; high-throughput detection of small molecules and nucleic acids; single enzyme activity

Special Issue Information

Dear Colleagues,

The recent implementation of state-of-the-art single-molecule techniques in cross-disciplinary science has transformed our understanding of biology and physics of life. Single-molecule techniques are the most desirable tool kits in many aspects of research today, not only to answer fundamental questions but also to enable high-sensitivity detection of small molecules and biomarkers. With their ever-improving spatial and temporal resolution, single-molecule techniques such as fluorescence microscopy, optical tweezers, and atomic force microscopy (AFM) have enabled single-molecule analysis of increasingly complex systems, such as live cells. Not amenable by bulk biochemical methods—because of averaging over large populations of molecules—single-molecule studies are significant to map reaction trajectories of individual molecules without the need for synchronization and to identify properties displayed by various molecular subpopulations. In addition, contrary to traditional ensemble methods that rely mainly in or close to the state of equilibrium, single-molecule techniques such as optical tweezers and AFM can illuminate the mechanism of biomolecular mechanics under strained conditions.

In this Special Issue on “Single-Molecule Techniques”, we welcome both original research and review articles as well as protocols pertaining to current applications of single-molecule techniques in many different areas of scientific inquiry including structural studies, DNA- as well as protein-DNA biophysics, enzymology, chemical biology, medicinal chemistry, nanotechnology and many more. In addition, we aim to present exemplary research findings from single-molecule studies.

Dr. Soma Dhakal
Guest Editor

Manuscript Submission Information

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Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) is waived for well-prepared manuscripts submitted to this issue. Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Single-molecule
  • Fluorescence Microscopy
  • Fluorescence resonance energy transfer (FRET)
  • Optical Tweezers
  • Atomic Force Microscopy (AFM)
  • Nanotechnology
  • Mechanical manipulation
  • Detection
  • DNA
  • Protein

Published Papers (5 papers)

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Review

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Open AccessReview
A Brief History of Single-Particle Tracking of the Epidermal Growth Factor Receptor
Methods Protoc. 2019, 2(1), 12; https://doi.org/10.3390/mps2010012
Received: 16 November 2018 / Revised: 21 January 2019 / Accepted: 21 January 2019 / Published: 30 January 2019
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Abstract
Single-particle tracking (SPT) has been used and developed over the last 25 years as a method to investigate molecular dynamics, structure, interactions, and function in the cellular context. SPT is able to show how fast and how far individual molecules move, identify different [...] Read more.
Single-particle tracking (SPT) has been used and developed over the last 25 years as a method to investigate molecular dynamics, structure, interactions, and function in the cellular context. SPT is able to show how fast and how far individual molecules move, identify different dynamic populations, measure the duration and strength of intermolecular interactions, and map out structures on the nanoscale in cells. In combination with other techniques such as macromolecular crystallography and molecular dynamics simulation, it allows us to build models of complex structures, and develop and test hypotheses of how these complexes perform their biological roles in health as well as in disease states. Here, we use the example of the epidermal growth factor receptor (EGFR), which has been studied extensively by SPT, demonstrating how the method has been used to increase our understanding of the receptor’s organization and function, including its interaction with the plasma membrane, its activation, clustering, and oligomerization, and the role of other receptors and endocytosis. The examples shown demonstrate how SPT might be employed in the investigation of other biomolecules and systems. Full article
(This article belongs to the Special Issue Single-Molecule Techniques)
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Other

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Open AccessProtocol
Using Single-Molecule Chemo-Mechanical Unfolding to Simultaneously Probe Multiple Structural Parameters in Protein Folding
Methods Protoc. 2019, 2(2), 32; https://doi.org/10.3390/mps2020032
Received: 11 March 2019 / Revised: 10 April 2019 / Accepted: 15 April 2019 / Published: 20 April 2019
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Abstract
While single-molecule force spectroscopy has greatly advanced the study of protein folding, there are limitations to what can be learned from studying the effect of force alone. We developed a novel technique, chemo-mechanical unfolding, that combines multiple perturbants—force and chemical denaturant—to more fully [...] Read more.
While single-molecule force spectroscopy has greatly advanced the study of protein folding, there are limitations to what can be learned from studying the effect of force alone. We developed a novel technique, chemo-mechanical unfolding, that combines multiple perturbants—force and chemical denaturant—to more fully characterize the folding process by simultaneously probing multiple structural parameters—the change in end-to-end distance, and solvent accessible surface area. Here, we describe the theoretical background, experimental design, and data analysis for chemo-mechanical unfolding experiments probing protein folding thermodynamics and kinetics. This technique has been applied to characterize parallel protein folding pathways, the protein denatured state, protein folding on the ribosome, and protein folding intermediates. Full article
(This article belongs to the Special Issue Single-Molecule Techniques)
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Open AccessProtocol
AFM-Based Force Spectroscopy Guided by Recognition Imaging: A New Mode for Mapping and Studying Interaction Sites at Low Lateral Density
Methods Protoc. 2019, 2(1), 6; https://doi.org/10.3390/mps2010006
Received: 19 October 2018 / Revised: 7 December 2018 / Accepted: 3 January 2019 / Published: 8 January 2019
Cited by 1 | PDF Full-text (3490 KB) | HTML Full-text | XML Full-text
Abstract
Ligand binding to receptors is one of the most important regulatory elements in biology as it is the initiating step in signaling pathways and cascades. Thus, precisely localizing binding sites and measuring interaction forces between cognate receptor–ligand pairs leads to new insights into [...] Read more.
Ligand binding to receptors is one of the most important regulatory elements in biology as it is the initiating step in signaling pathways and cascades. Thus, precisely localizing binding sites and measuring interaction forces between cognate receptor–ligand pairs leads to new insights into the molecular recognition involved in these processes. Here we present a detailed protocol about applying a technique, which combines atomic force microscopy (AFM)-based recognition imaging and force spectroscopy for studying the interaction between (membrane) receptors and ligands on the single molecule level. This method allows for the selection of a single receptor molecule reconstituted into a supported lipid membrane at low density, with the subsequent quantification of the receptor–ligand unbinding force. Based on AFM tapping mode, a cantilever tip carrying a ligand molecule is oscillated across a membrane. Topography and recognition images of reconstituted receptors are recorded simultaneously by analyzing the downward and upward parts of the oscillation, respectively. Functional receptor molecules are selected from the recognition image with nanometer resolution before the AFM is switched to the force spectroscopy mode, using positional feedback control. The combined mode allows for dynamic force probing on different pre-selected molecules. This strategy results in higher throughput when compared with force mapping. Applied to two different receptor–ligand pairs, we validated the presented new mode. Full article
(This article belongs to the Special Issue Single-Molecule Techniques)
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Open AccessBenchmark
Optical Trapping and Manipulation of Superparamagnetic Beads Using Annular-Shaped Beams
Methods Protoc. 2018, 1(4), 44; https://doi.org/10.3390/mps1040044
Received: 9 October 2018 / Revised: 12 November 2018 / Accepted: 14 November 2018 / Published: 20 November 2018
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Abstract
We propose an optical tweezers setup based on an annular-shaped laser beam that is efficient to trap 2.8 μm-diameter superparamagnetic particles. The optical trapping of such particles was fully characterized, and a direct absolute comparison with a geometrical optics model was performed. [...] Read more.
We propose an optical tweezers setup based on an annular-shaped laser beam that is efficient to trap 2.8 μ m-diameter superparamagnetic particles. The optical trapping of such particles was fully characterized, and a direct absolute comparison with a geometrical optics model was performed. With this comparison, we were able to show that light absorption by the superparamagnetic particles is negligible for our annular beam tweezers, differing from the case of conventional Gaussian beam tweezers, in which laser absorption by the beads makes stable trapping difficult. In addition, the trap stiffness of the annular beam tweezers increases with the laser power and with the bead distance from the coverslip surface. While this first result is expected and similar to that achieved for conventional Gaussian tweezers, which use ordinary dielectric beads, the second result is quite surprising and different from the ordinary case, suggesting that spherical aberration is much less important in our annular beam geometry. The results obtained here provide new insights into the development of hybrid optomagnetic tweezers, which can apply simultaneously optical and magnetic forces on the same particles. Full article
(This article belongs to the Special Issue Single-Molecule Techniques)
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Open AccessProtocol
Build Your Own Microscope: Step-By-Step Guide for Building a Prism-Based TIRF Microscope
Methods Protoc. 2018, 1(4), 40; https://doi.org/10.3390/mps1040040
Received: 29 September 2018 / Revised: 31 October 2018 / Accepted: 31 October 2018 / Published: 3 November 2018
Cited by 1 | PDF Full-text (5270 KB) | HTML Full-text | XML Full-text
Abstract
Prism-based total internal reflection fluorescence (pTIRF) microscopy is one of the most widely used techniques for the single molecule analysis of a vast range of samples including biomolecules, nanostructures, and cells, to name a few. It allows for excitation of surface bound molecules/particles/quantum [...] Read more.
Prism-based total internal reflection fluorescence (pTIRF) microscopy is one of the most widely used techniques for the single molecule analysis of a vast range of samples including biomolecules, nanostructures, and cells, to name a few. It allows for excitation of surface bound molecules/particles/quantum dots via evanescent field of a confined region of space, which is beneficial not only for single molecule detection but also for analysis of single molecule dynamics and for acquiring kinetics data. However, there is neither a commercial microscope available for purchase nor a detailed guide dedicated for building this microscope. Thus far, pTIRF microscopes are custom-built with the use of a commercially available inverted microscope, which requires high level of expertise in selecting and handling sophisticated instrument-parts. To directly address this technology gap, here we describe a step-by-step guide on how to build and characterize a pTIRF microscope for in vitro single-molecule imaging, nanostructure analysis and other life sciences research. Full article
(This article belongs to the Special Issue Single-Molecule Techniques)
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