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Special Issue "Single Molecule Techniques"

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A special issue of Molecules (ISSN 1420-3049). This special issue belongs to the section "Molecular Diversity".

Deadline for manuscript submissions: closed (15 June 2014)

Special Issue Editor

Guest Editor
Dr. Hans-Heiner Gorris (Website)

Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Universitätsstrasse 31, 93040 Regensburg, Germany
Interests: single molecule analysis in femtoliter arrays; enzyme kinetics; ultrasensitive and background-free detection of analytes; photon-upconverting nanoparticles

Special Issue Information

Dear Colleagues,

Single molecule experiments have unraveled processes that were previously concealed in bulk experiments because they were not synchronized or even heterogeneously distributed among individual molecules in a population. New insights into the function, structure and interactions of individual molecules have been driven by technological advances in single molecule manipulation and detection. Fluorescence spectroscopy is one of the most powerful single molecule techniques because it has a high spatial and temporal resolution and is very sensitive, as long as the background can be efficiently suppressed.

In biochemistry/biophysics, transitions or differences in the conformation of proteins have been observed as dynamic or static heterogeneity. Labeling of proteins and DNA with donor-quencher pairs has enabled the detection of binding interactions by fluorescence resonance energy transfer (FRET). Site-specific labeling of protein subunits has enabled the use of FRET as a nanoscale ruler for measuring conformational transitions. On the other hand, fluorogenic substrates can be employed for investigating the catalytic activity of single enzyme molecules. The analysis of single molecule binding and transition events has entailed a shift from a kinetic to a stochastic perspective on biochemical processes.

This Special Issue of Molecules covers all aspects related to the development and the application of "Single Molecule Techniques". Not only are techniques based on fluorescence spectroscopy welcome but also those based on atomic force spectroscopy, scanning-tunneling electron microscopy or others. It is a pleasure to invite original research as well as review articles that describe and discuss technical developments in the detection and manipulation of single molecules as well as their applications.

Dr. Hans-H. Gorris
Guest Editor

Submission

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. Papers will be published continuously (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are refereed through a peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Molecules is an international peer-reviewed Open Access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 1800 CHF (Swiss Francs).


Keywords

  • biochemistry/biophysics
  • conformational dynamics
  • DNA
  • dynamic/static heterogeneity
  • fluorescence resonance energy transfer (FRET)
  • fluorescence spectroscopy
  • proteins
  • single molecules
  • super-resolution microscopy

Published Papers (13 papers)

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Editorial

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Open AccessEditorial Special Issue: Single Molecule Techniques
Molecules 2015, 20(5), 7772-7774; doi:10.3390/molecules20057772
Received: 24 April 2015 / Accepted: 27 April 2015 / Published: 28 April 2015
PDF Full-text (609 KB) | HTML Full-text | XML Full-text
Abstract
Technological advances in the detection and manipulation of single molecules have enabled new insights into the function, structure and interactions of biomolecules. This Special Issue was launched to account for the rapid progress in the field of “Single Molecule Techniques”. Four original [...] Read more.
Technological advances in the detection and manipulation of single molecules have enabled new insights into the function, structure and interactions of biomolecules. This Special Issue was launched to account for the rapid progress in the field of “Single Molecule Techniques”. Four original research articles and seven review articles provide an introduction, as well as an in-depth discussion, of technical developments that are indispensable for the characterization of individual biomolecules. Fluorescence microscopy takes center stage in this Special Issue because it is one of the most sensitive and flexible techniques, which has been adapted in many variations to the specific demands of single molecule analysis. Two additional articles are dedicated to single molecule detection based on atomic force microscopy. Full article
(This article belongs to the Special Issue Single Molecule Techniques)

Research

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Open AccessArticle Inter-Dye Distance Distributions Studied by a Combination of Single-Molecule FRET-Filtered Lifetime Measurements and a Weighted Accessible Volume (wAV) Algorithm
Molecules 2014, 19(12), 19269-19291; doi:10.3390/molecules191219269
Received: 23 August 2014 / Revised: 3 November 2014 / Accepted: 6 November 2014 / Published: 25 November 2014
Cited by 3 | PDF Full-text (1981 KB) | HTML Full-text | XML Full-text
Abstract
Förster resonance energy transfer (FRET) is an important tool for studying the structural and dynamical properties of biomolecules. The fact that both the internal dynamics of the biomolecule and the movements of the biomolecule-attached dyes can occur on similar timescales of nanoseconds [...] Read more.
Förster resonance energy transfer (FRET) is an important tool for studying the structural and dynamical properties of biomolecules. The fact that both the internal dynamics of the biomolecule and the movements of the biomolecule-attached dyes can occur on similar timescales of nanoseconds is an inherent problem in FRET studies. By performing single-molecule FRET-filtered lifetime measurements, we are able to characterize the amplitude of the motions of fluorescent probes attached to double-stranded DNA standards by means of flexible linkers. With respect to previously proposed experimental approaches, we improved the precision and the accuracy of the inter-dye distance distribution parameters by filtering out the donor-only population with pulsed interleaved excitation. A coarse-grained model is employed to reproduce the experimentally determined inter-dye distance distributions. This approach can easily be extended to intrinsically flexible proteins allowing, under certain conditions, to decouple the macromolecule amplitude of motions from the contribution of the dye linkers. Full article
(This article belongs to the Special Issue Single Molecule Techniques)
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Open AccessArticle Data-Driven Techniques for Detecting Dynamical State Changes in Noisily Measured 3D Single-Molecule Trajectories
Molecules 2014, 19(11), 18381-18398; doi:10.3390/molecules191118381
Received: 1 September 2014 / Revised: 28 October 2014 / Accepted: 29 October 2014 / Published: 12 November 2014
Cited by 4 | PDF Full-text (3710 KB) | HTML Full-text | XML Full-text | Correction
Abstract
Optical microscopes and nanoscale probes (AFM, optical tweezers, etc.) afford researchers tools capable of quantitatively exploring how molecules interact with one another in live cells. The analysis of in vivo single-molecule experimental data faces numerous challenges due to the complex, crowded, [...] Read more.
Optical microscopes and nanoscale probes (AFM, optical tweezers, etc.) afford researchers tools capable of quantitatively exploring how molecules interact with one another in live cells. The analysis of in vivo single-molecule experimental data faces numerous challenges due to the complex, crowded, and time changing environments associated with live cells. Fluctuations and spatially varying systematic forces experienced by molecules change over time; these changes are obscured by “measurement noise” introduced by the experimental probe monitoring the system. In this article, we demonstrate how the Hierarchical Dirichlet Process Switching Linear Dynamical System (HDP-SLDS) of Fox et al. [IEEE Transactions on Signal Processing 59] can be used to detect both subtle and abrupt state changes in time series containing “thermal” and “measurement” noise. The approach accounts for temporal dependencies induced by random and “systematic overdamped” forces. The technique does not require one to subjectively select the number of “hidden states” underlying a trajectory in an a priori fashion. The number of hidden states is simultaneously inferred along with change points and parameters characterizing molecular motion in a data-driven fashion. We use large scale simulations to study and compare the new approach to state-of-the-art Hidden Markov Modeling techniques. Simulations mimicking single particle tracking (SPT) experiments are the focus of this study. Full article
(This article belongs to the Special Issue Single Molecule Techniques)
Open AccessArticle Different Fluorophore Labeling Strategies and Designs Affect Millisecond Kinetics of DNA Hairpins
Molecules 2014, 19(9), 13735-13754; doi:10.3390/molecules190913735
Received: 18 July 2014 / Revised: 21 August 2014 / Accepted: 26 August 2014 / Published: 3 September 2014
Cited by 4 | PDF Full-text (1621 KB) | HTML Full-text | XML Full-text
Abstract
Changes in molecular conformations are one of the major driving forces of complex biological processes. Many studies based on single-molecule techniques have shed light on conformational dynamics and contributed to a better understanding of living matter. In particular, single-molecule FRET experiments have [...] Read more.
Changes in molecular conformations are one of the major driving forces of complex biological processes. Many studies based on single-molecule techniques have shed light on conformational dynamics and contributed to a better understanding of living matter. In particular, single-molecule FRET experiments have revealed unprecedented information at various time scales varying from milliseconds to seconds. The choice and the attachment of fluorophores is a pivotal requirement for single-molecule FRET experiments. One particularly well-studied millisecond conformational change is the opening and closing of DNA hairpin structures. In this study, we addressed the influence of base- and terminal-labeled fluorophores as well as the fluorophore DNA interactions on the extracted kinetic information of the DNA hairpin. Gibbs free energies varied from ∆G0 = −3.6 kJ/mol to ∆G0 = −0.2 kJ/mol for the identical DNA hairpin modifying only the labeling scheme and design of the DNA sample. In general, the base-labeled DNA hairpin is significantly destabilized compared to the terminal-labeled DNA hairpin and fluorophore DNA interactions additionally stabilize the closed state of the DNA hairpin. Careful controls and variations of fluorophore attachment chemistry are essential for a mostly undisturbed measurement of the underlying energy landscape of biomolecules. Full article
(This article belongs to the Special Issue Single Molecule Techniques)
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Open AccessArticle pH-Dependent Deformations of the Energy Landscape of Avidin-like Proteins Investigated by Single Molecule Force Spectroscopy
Molecules 2014, 19(8), 12531-12546; doi:10.3390/molecules190812531
Received: 16 June 2014 / Revised: 31 July 2014 / Accepted: 7 August 2014 / Published: 18 August 2014
Cited by 2 | PDF Full-text (1420 KB) | HTML Full-text | XML Full-text
Abstract
Avidin and avidin-like proteins are widely used in numerous techniques since the avidin-biotin interaction is known to be very robust and reliable. Within this study, we investigated this bond at the molecular level under harsh conditions ranging from very low to very [...] Read more.
Avidin and avidin-like proteins are widely used in numerous techniques since the avidin-biotin interaction is known to be very robust and reliable. Within this study, we investigated this bond at the molecular level under harsh conditions ranging from very low to very high pH values. We compared avidin with streptavidin and a recently developed avidin-based mutant, chimeric avidin. To gain insights of the energy landscape of these interactions we used a single molecule approach and performed the Single Molecule Force Spectroscopy atomic force microscopy technique. There, the ligand (biotin) is covalently coupled to a sharp AFM tip via a distensible hetero-bi-functional crosslinker, whereas the receptor of interest is immobilized on the probe surface. Receptor-ligand complexes are formed and ruptured by repeatedly approaching and withdrawing the tip from the surface. Varying both pulling velocity and pH value, we could determine changes of the energy landscape of the complexes. Our results clearly demonstrate that avidin, streptavidin and chimeric avidin are stable over a wide pH range although we could identify differences at the outer pH range. Taking this into account, they can be used in a broad range of applications, like surface sensors at extreme pH values. Full article
(This article belongs to the Special Issue Single Molecule Techniques)

Review

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Open AccessReview Shedding Light on Protein Folding, Structural and Functional Dynamics by Single Molecule Studies
Molecules 2014, 19(12), 19407-19434; doi:10.3390/molecules191219407
Received: 30 July 2014 / Revised: 7 November 2014 / Accepted: 12 November 2014 / Published: 25 November 2014
Cited by 6 | PDF Full-text (3574 KB) | HTML Full-text | XML Full-text
Abstract
The advent of advanced single molecule measurements unveiled a great wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways unattainable by conventional bulk assays. Equipped with the ability to record distribution of behaviors rather than the mean [...] Read more.
The advent of advanced single molecule measurements unveiled a great wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways unattainable by conventional bulk assays. Equipped with the ability to record distribution of behaviors rather than the mean property of a population, single molecule measurements offer observation and quantification of the abundance, lifetime and function of multiple protein states. They also permit the direct observation of the transient and rarely populated intermediates in the energy landscape that are typically averaged out in non-synchronized ensemble measurements. Single molecule studies have thus provided novel insights about how the dynamic sampling of the free energy landscape dictates all aspects of protein behavior; from its folding to function. Here we will survey some of the state of the art contributions in deciphering mechanisms that underlie protein folding, structural and functional dynamics by single molecule fluorescence microscopy techniques. We will discuss a few selected examples highlighting the power of the emerging techniques and finally discuss the future improvements and directions. Full article
(This article belongs to the Special Issue Single Molecule Techniques)
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Open AccessReview A Starting Point for Fluorescence-Based Single-Molecule Measurements in Biomolecular Research
Molecules 2014, 19(10), 15824-15865; doi:10.3390/molecules191015824
Received: 21 July 2014 / Revised: 17 September 2014 / Accepted: 17 September 2014 / Published: 30 September 2014
Cited by 13 | PDF Full-text (2951 KB) | HTML Full-text | XML Full-text
Abstract
Single-molecule fluorescence techniques are ideally suited to provide information about the structure-function-dynamics relationship of a biomolecule as static and dynamic heterogeneity can be easily detected. However, what type of single-molecule fluorescence technique is suited for which kind of biological question and what [...] Read more.
Single-molecule fluorescence techniques are ideally suited to provide information about the structure-function-dynamics relationship of a biomolecule as static and dynamic heterogeneity can be easily detected. However, what type of single-molecule fluorescence technique is suited for which kind of biological question and what are the obstacles on the way to a successful single-molecule microscopy experiment? In this review, we provide practical insights into fluorescence-based single-molecule experiments aiming for scientists who wish to take their experiments to the single-molecule level. We especially focus on fluorescence resonance energy transfer (FRET) experiments as these are a widely employed tool for the investigation of biomolecular mechanisms. We will guide the reader through the most critical steps that determine the success and quality of diffusion-based confocal and immobilization-based total internal reflection fluorescence microscopy. We discuss the specific chemical and photophysical requirements that make fluorescent dyes suitable for single-molecule fluorescence experiments. Most importantly, we review recently emerged photoprotection systems as well as passivation and immobilization strategies that enable the observation of fluorescently labeled molecules under biocompatible conditions. Moreover, we discuss how the optical single-molecule toolkit has been extended in recent years to capture the physiological complexity of a cell making it even more relevant for biological research. Full article
(This article belongs to the Special Issue Single Molecule Techniques)
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Open AccessReview Enzyme Molecules in Solitary Confinement
Molecules 2014, 19(9), 14417-14445; doi:10.3390/molecules190914417
Received: 4 August 2014 / Revised: 3 September 2014 / Accepted: 3 September 2014 / Published: 12 September 2014
Cited by 7 | PDF Full-text (3050 KB) | HTML Full-text | XML Full-text
Abstract
Large arrays of homogeneous microwells each defining a femtoliter volume are a versatile platform for monitoring the substrate turnover of many individual enzyme molecules in parallel. The high degree of parallelization enables the analysis of a statistically representative enzyme population. Enclosing individual [...] Read more.
Large arrays of homogeneous microwells each defining a femtoliter volume are a versatile platform for monitoring the substrate turnover of many individual enzyme molecules in parallel. The high degree of parallelization enables the analysis of a statistically representative enzyme population. Enclosing individual enzyme molecules in microwells does not require any surface immobilization step and enables the kinetic investigation of enzymes free in solution. This review describes various microwell array formats and explores their applications for the detection and investigation of single enzyme molecules. The development of new fabrication techniques and sensitive detection methods drives the field of single molecule enzymology. Here, we introduce recent progress in single enzyme molecule analysis in microwell arrays and discuss the challenges and opportunities. Full article
(This article belongs to the Special Issue Single Molecule Techniques)
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Open AccessReview Protein Expression Analyses at the Single Cell Level
Molecules 2014, 19(9), 13932-13947; doi:10.3390/molecules190913932
Received: 30 June 2014 / Revised: 13 August 2014 / Accepted: 29 August 2014 / Published: 5 September 2014
Cited by 3 | PDF Full-text (2164 KB) | HTML Full-text | XML Full-text
Abstract
The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, [...] Read more.
The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level. Full article
(This article belongs to the Special Issue Single Molecule Techniques)
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Open AccessReview Molecular Processes Studied at a Single-Molecule Level Using DNA Origami Nanostructures and Atomic Force Microscopy
Molecules 2014, 19(9), 13803-13823; doi:10.3390/molecules190913803
Received: 23 July 2014 / Revised: 21 August 2014 / Accepted: 29 August 2014 / Published: 3 September 2014
Cited by 10 | PDF Full-text (14040 KB) | HTML Full-text | XML Full-text
Abstract
DNA origami nanostructures allow for the arrangement of different functionalities such as proteins, specific DNA structures, nanoparticles, and various chemical modifications with unprecedented precision. The arranged functional entities can be visualized by atomic force microscopy (AFM) which enables the study of molecular [...] Read more.
DNA origami nanostructures allow for the arrangement of different functionalities such as proteins, specific DNA structures, nanoparticles, and various chemical modifications with unprecedented precision. The arranged functional entities can be visualized by atomic force microscopy (AFM) which enables the study of molecular processes at a single-molecular level. Examples comprise the investigation of chemical reactions, electron-induced bond breaking, enzymatic binding and cleavage events, and conformational transitions in DNA. In this paper, we provide an overview of the advances achieved in the field of single-molecule investigations by applying atomic force microscopy to functionalized DNA origami substrates. Full article
(This article belongs to the Special Issue Single Molecule Techniques)
Open AccessReview Imaging Live Cells at the Nanometer-Scale with Single-Molecule Microscopy: Obstacles and Achievements in Experiment Optimization for Microbiology
Molecules 2014, 19(8), 12116-12149; doi:10.3390/molecules190812116
Received: 24 June 2014 / Revised: 1 August 2014 / Accepted: 1 August 2014 / Published: 13 August 2014
Cited by 19 | PDF Full-text (6136 KB) | HTML Full-text | XML Full-text
Abstract
Single-molecule fluorescence microscopy enables biological investigations inside living cells to achieve millisecond- and nanometer-scale resolution. Although single-molecule-based methods are becoming increasingly accessible to non-experts, optimizing new single-molecule experiments can be challenging, in particular when super-resolution imaging and tracking are applied to live [...] Read more.
Single-molecule fluorescence microscopy enables biological investigations inside living cells to achieve millisecond- and nanometer-scale resolution. Although single-molecule-based methods are becoming increasingly accessible to non-experts, optimizing new single-molecule experiments can be challenging, in particular when super-resolution imaging and tracking are applied to live cells. In this review, we summarize common obstacles to live-cell single-molecule microscopy and describe the methods we have developed and applied to overcome these challenges in live bacteria. We examine the choice of fluorophore and labeling scheme, approaches to achieving single-molecule levels of fluorescence, considerations for maintaining cell viability, and strategies for detecting single-molecule signals in the presence of noise and sample drift. We also discuss methods for analyzing single-molecule trajectories and the challenges presented by the finite size of a bacterial cell and the curvature of the bacterial membrane. Full article
(This article belongs to the Special Issue Single Molecule Techniques)
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Open AccessReview Tracking Electrons in Biological Macromolecules: From Ensemble to Single Molecule
Molecules 2014, 19(8), 11660-11678; doi:10.3390/molecules190811660
Received: 4 July 2014 / Revised: 23 July 2014 / Accepted: 25 July 2014 / Published: 6 August 2014
Cited by 2 | PDF Full-text (7038 KB) | HTML Full-text | XML Full-text
Abstract
Nature utilizes oxido-reductases to cater to the energy demands of most biochemical processes in respiratory species. Oxido-reductases are capable of meeting this challenge by utilizing redox active sites, often containing transition metal ions, which facilitate movement and relocation of electrons/protons to create [...] Read more.
Nature utilizes oxido-reductases to cater to the energy demands of most biochemical processes in respiratory species. Oxido-reductases are capable of meeting this challenge by utilizing redox active sites, often containing transition metal ions, which facilitate movement and relocation of electrons/protons to create a potential gradient that is used to energize redox reactions. There has been a consistent struggle by researchers to estimate the electron transfer rate constants in physiologically relevant processes. This review provides a brief background on the measurements of electron transfer rates in biological molecules, in particular Cu-containing enzymes, and highlights the recent advances in monitoring these electron transfer events at the single molecule level or better to say, at the individual event level. Full article
(This article belongs to the Special Issue Single Molecule Techniques)
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Other

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Open AccessCorrection Correction: Calderon, C.P. Data-Driven Techniques for Detecting Dynamical State Changes in Noisily Measured 3D Single-Molecule Trajectories. Molecules 19, 18381-18398
Molecules 2015, 20(2), 2828-2830; doi:10.3390/molecules20022828
Received: 20 January 2015 / Accepted: 4 February 2015 / Published: 9 February 2015
PDF Full-text (768 KB) | HTML Full-text | XML Full-text
Abstract The author wishes to make the following corrections to paper [1] (doi:10.3390/molecules191118381, website: http://www.mdpi.com/1420-3049/19/11/18381): Full article
(This article belongs to the Special Issue Single Molecule Techniques)

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