scholarly journals The more the merrier: high-throughput single-molecule techniques

2017 ◽  
Vol 45 (3) ◽  
pp. 759-769 ◽  
Author(s):  
Flynn R. Hill ◽  
Enrico Monachino ◽  
Antoine M. van Oijen
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Zero Mode ◽  
Magnetic Tweezers ◽  
Ease Of Use ◽  
Single Molecule Fluorescence ◽  
Significant Challenge ◽  
Large Numbers ◽  
Very High

The single-molecule approach seeks to understand molecular mechanisms by observing biomolecular processes at the level of individual molecules. These methods have led to a developing understanding that for many processes, a diversity of behaviours will be observed, representing a multitude of pathways. This realisation necessitates that an adequate number of observations are recorded to fully characterise this diversity. The requirement for large numbers of observations to adequately sample distributions, subpopulations, and rare events presents a significant challenge for single-molecule techniques, which by their nature do not typically provide very high throughput. This review will discuss many developing techniques which address this issue by combining nanolithographic approaches, such as zero-mode waveguides and DNA curtains, with single-molecule fluorescence microscopy, and by drastically increasing throughput of force-based approaches such as magnetic tweezers and laminar-flow techniques. These methods not only allow the collection of large volumes of single-molecule data in single experiments, but have also made improvements to ease-of-use, accessibility, and automation of data analysis.

scholarly journals Single-molecule fluorescence imaging of processive myosin with enhanced background suppression using linear zero-mode waveguides (ZMWs) and convex lens induced confinement (CLIC)

2013 ◽  
Vol 21 (1) ◽  
pp. 1189 ◽  
Author(s):  
Mary Williard Elting ◽  
Sabrina R. Leslie ◽  
L. Stirling Churchman ◽  
Jonas Korlach ◽  
Christopher M. J. McFaul ◽  
...  
Keyword(s):  
Fluorescence Imaging ◽  
Single Molecule ◽  
Zero Mode ◽  
Background Suppression ◽  
Single Molecule Fluorescence ◽  
Convex Lens

Single-molecule fluorescence imaging of kinesin using linear zero-mode waveguides

2016 ◽  
Author(s):  
Yuki Morita ◽  
Kazuya Fujimoto ◽  
Ryota Iino ◽  
Michio Tomishige ◽  
Hirofumi Shintaku ◽  
...  
Keyword(s):  
Fluorescence Imaging ◽  
Single Molecule ◽  
Zero Mode ◽  
Single Molecule Fluorescence

scholarly journals High throughput mechanobiology: Force modulation of ensemble biochemical and cell-based assays

2020 ◽  
Author(s):  
Ália dos Santos ◽  
Natalia Fili ◽  
David S. Pearson ◽  
Yukti Hari-Gupta ◽  
Christopher P. Toseland
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Receptor Activation ◽  
Magnetic Tweezers ◽  
Live Cells ◽  
Mechanical Stimuli ◽  
Cellular Assays ◽  
Novel Approach ◽  
Microplate Reader

ABSTRACTMechanobiology is focused on how the physical forces and the mechanical properties of proteins, cells and tissues contribute to physiology and disease. While the response of proteins and cells to mechanical stimuli is critical for function, the tools to probe these activities are typically restricted to single molecule manipulations. Here, we have developed a novel microplate reader assay to encompass mechanical measurements with ensemble biochemical and cellular assays, using a microplate lid modified with magnets. This configuration enables multiple static magnetic tweezers to function simultaneously across the microplate, thereby greatly increasing throughput. The broad applicability and versatility of our approach has been demonstrated through in vitro force-induced enzymatic activity and conformation changes, along with force-induced receptor activation and their downstream signalling pathways in live cells. Overall, our methodology allows for the first-time ensemble biochemical and cell-based assays to be performed under force, in high throughput format. This novel approach would substantially add to the mechano-biological toolbox and increase the availability of mechanobiology measurements.

scholarly journals Temperature controlled high-throughput magnetic tweezers show striking difference in activation energies of replicating viral RNA-dependent RNA polymerases

2020 ◽  
Author(s):  
M. Seifert ◽  
P. van Nies ◽  
F.S. Papini ◽  
J.J. Arnold ◽  
M.M. Poranen ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Rna Virus ◽  
Magnetic Tweezers ◽  
Activation Energies ◽  
Viral Rna ◽  
Controlled Study ◽  
Striking Difference ◽  
Genome Replication ◽  
Temperature Controlled

AbstractRNA virus survival depends on efficient viral genome replication, which is performed by the viral RNA dependent RNA polymerase (RdRp). The recent development of high throughput magnetic tweezers has enabled the simultaneous observation of dozens of viral RdRp elongation traces on kilobases long templates, and this has shown that RdRp nucleotide addition kinetics is stochastically interrupted by rare pauses of 1-1000 s duration, of which the short-lived ones (1-10 s) are the temporal signature of a low fidelity catalytic pathway. We present a simple and precise temperature controlled system for magnetic tweezers to characterize the replication kinetics temperature dependence between 25°C and 45°C of RdRps from three RNA viruses, i.e. the double-stranded RNA bacteriophage Φ6, and the positive-sense single-stranded RNA poliovirus (PV) and human rhinovirus C (HRV-C). We found that Φ6 RdRp is largely temperature insensitive, while PV and HRV-C RdRps replication kinetics are activated by temperature. Furthermore, the activation energies we measured for PV RdRp catalytic state corroborate previous estimations from ensemble pre-steady state kinetic studies, further confirming the catalytic origin of the short pauses and their link to temperature independent RdRp fidelity. This work will enable future temperature controlled study of biomolecular complex at the single molecule level.

scholarly journals Nanoaperture fabrication via colloidal lithography for single molecule fluorescence imaging

2019 ◽  
Author(s):  
Ryan M. Jamiolkowski ◽  
Kevin Y Chen ◽  
Shane A. Fiorenza ◽  
Alyssa M. Tate ◽  
Shawn H. Pfeil ◽  
...  
Keyword(s):  
Single Molecule ◽  
Self Assembly ◽  
Zero Mode ◽  
Thin Metal Film ◽  
Polystyrene Microspheres ◽  
Single Molecule Fluorescence ◽  
Fluorescence Studies ◽  
Observation Volume ◽  
Sophisticated Equipment ◽  
Sub Wavelength

AbstractIn single molecule fluorescence studies, background emission from labeled substrates often restricts their concentrations to non-physiological nanomolar values. One approach to address this challenge is the use of zero-mode waveguides (ZMWs), nanoscale holes in a thin metal film that physically and optically confine the observation volume allowing much higher concentrations of fluorescent substrates. Standard fabrication of ZMWs utilizes slow and costly E-beam nano-lithography. Herein, ZMWs are made using a self-assembled mask of polystyrene microspheres, enabling fabrication of thousands of ZMWs in parallel without sophisticated equipment. Polystyrene 1 μm dia. microbeads self-assemble on a glass slide into a hexagonal array, forming a mask for the deposition of metallic posts in the inter-bead interstices. The width of those interstices (and subsequent posts) is adjusted within 100-300 nm by partially fusing the beads at the polystyrene glass transition temperature. The beads are dissolved in toluene, aluminum or gold cladding is deposited around the posts, and those are dissolved, leaving behind an array ZMWs. Parameter optimization and the performance of the ZMWs are presented. By using colloidal self-assembly, typical laboratories can make use of sub-wavelength ZMW technology avoiding the availability and expense of sophisticated clean-room environments and equipment.

scholarly journals Single-molecule assay for proteolytic susceptibility using centrifuge force microscopy:force-induced destabilization of collagen‘s triple helix

2017 ◽  
Author(s):  
Michael W.H. Kirkness ◽  
Nancy R. Forde
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Triple Helix ◽  
Proteolytic Cleavage ◽  
Ease Of Use ◽  
Enzymatic Assay ◽  
Biomolecular Structure ◽  
Collagen Molecules ◽  
Time Readout

Force plays a key role in regulating dynamics of biomolecular structure and interactions, yet techniques are lacking to manipulate and continuously read out this response with high throughput. We present an enzymatic assay for force-dependent accessibility of structure that makes use of a wireless mini-radio centrifuge force microscope (MR.CFM) to provide a real-time readout of kinetics. The microscope is designed for ease of use, fits in a standard centrifuge bucket, and offers high-throughput, video-rate readout of individual proteolytic cleavage events. Proteolysis measurements on thousands of tethered collagen molecules show a load-enhanced trypsin sensitivity, indicating destabilization of the triple helix.

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