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.

Highly Sensitive Detection of Paraquat with Pillar[5]arene as an aptamer in α-Hemolysin Nanopore

2021 ◽  
Author(s):  
Xiaojia Jiang ◽  
Mingsong Zang ◽  
Fei Li ◽  
Chunxi Hou ◽  
Quan Luo ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Single Molecule Detection ◽  
Sensitive Detection ◽  
High Throughput Analysis ◽  
Throughput Analysis ◽  
The Real ◽  
Highly Sensitive ◽  
Highly Sensitive Detection

Biological nanopore-based techniques have attracted more and more attention recently in the field of single-molecule detection, because they allow the real-time, sensitive, high-throughput analysis. Herein, we report an engineered biological...

scholarly journals Massively parallel single-molecule telomere length measurement with digital real-time PCR

2020 ◽  
Vol 6 (34) ◽  
pp. eabb7944 ◽  
Author(s):  
Yongqiang Luo ◽  
Ramya Viswanathan ◽  
Manoor Prakash Hande ◽  
Amos Hong Pheng Loh ◽  
Lih Feng Cheow
Keyword(s):  
Telomere Length ◽  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Real Time Pcr ◽  
Length Distribution ◽  
Telomere Maintenance ◽  
Massively Parallel ◽  
Primary Tumors ◽  
Absolute Length

Telomere length is a promising biomarker for age-associated diseases and cancer, but there are still substantial challenges to routine telomere analysis in clinics because of the lack of a simple and rapid yet scalable method for measurement. We developed the single telomere absolute-length rapid (STAR) assay, a novel high-throughput digital real-time PCR approach for rapidly measuring the absolute lengths and quantities of individual telomere molecules. We show that this technique provides the accuracy and sensitivity to uncover associations between telomere length distribution and telomere maintenance mechanisms in cancer cell lines and primary tumors. The results indicate that the STAR assay is a powerful tool to enable the use of telomere length distribution as a biomarker in disease and population-wide studies.

Nanoscale “Curtain Rods”: High-Throughput Tools for Studying DNA-Protein Interactions

2008 ◽  
Vol 1138 ◽  
Author(s):  
Teresa Fazio ◽  
Mari-Liis Visnapuu ◽  
Shalom J. Wind ◽  
Eric Greene
Keyword(s):  
Data Acquisition ◽  
Real Time ◽  
Lipid Bilayer ◽  
High Throughput ◽  
Single Molecule ◽  
Protein Interactions ◽  
Massively Parallel ◽  
Dna Interactions ◽  
Parallel Data ◽  
Protein Dna Interactions

AbstractIn this work, we combine nanoscale engineering with single-molecule biology to probe the biochemical interactions between individual proteins and DNA. This approach, a vast improvement over previous methods, constructs a platform to observe thousands of protein-DNA interactions in real time with unprecedented detail. A key challenge in these experiments involves collecting enough statistically relevant data in order to analyze reactions which are designed to be probed individually. “DNA curtains” are formed by flowing the DNA tethered to a lipid bilayer across nanopatterned barriers, facilitating massively parallel data acquisition.

scholarly journals 48-spot single-molecule FRET setup with periodic acceptor excitation

2017 ◽  
Author(s):  
Antonino Ingargiola ◽  
Maya Segal ◽  
Angelo Gulinatti ◽  
Ivan Rech ◽  
Ivan Labanca ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Conformational Changes ◽  
High Throughput Screening ◽  
Acquisition Time ◽  
Enzymatic Reactions ◽  
Single Molecule Fret ◽  
Array Detectors ◽  
Donor And Acceptor

Single-molecule FRET (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser exci-tation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hin-ders the ability to study kinetic phenomena in real-time.Here we demonstrate a high-throughput smFRET system which multiplexes acquisition by using 48 excitation spots and two 48-pixel SPAD array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly-labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

scholarly journals Massively multiplex single-molecule oligonucleosome footprinting

2020 ◽  
Author(s):  
Nour J Abdulhay ◽  
Colin P McNally ◽  
Laura J Hsieh ◽  
Sivakanthan Kasinathan ◽  
Aidan Keith ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Nucleosome Occupancy ◽  
Binding Motifs ◽  
Single Molecule Sequencing ◽  
Genome Wide ◽  
Sequencing Method ◽  
Transcription Factor Binding Motifs

ABSTRACTOur understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows unbiased classification of single-molecular ‘states’ of nucleosome occupancy on individual chromatin fibres. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at predicted transcription factor binding motifs, and across both active and silent human epigenomic domains. Our analyses suggest that chromatin is comprised of a diverse array of both regular and irregular single-molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying nucleosome organization at a previously intractable resolution, and offers up new avenues for modeling and visualizing higher-order chromatin structure.1-sentence summaryHigh-throughput single-molecule real-time footprinting of chromatin arrays reveals heterogeneous patterns of oligonucleosome occupancy.

scholarly journals High Throughput Random Mutagenesis and Single Molecule Real Time Sequencing of the Muscle Nicotinic Acetylcholine Receptor

PLoS ONE ◽  
2016 ◽  
Vol 11 (9) ◽  
pp. e0163129 ◽  
Author(s):  
Paul J. Groot-Kormelink ◽  
Sandrine Ferrand ◽  
Nicholas Kelley ◽  
Anke Bill ◽  
Felix Freuler ◽  
...  
Keyword(s):  
Real Time ◽  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
High Throughput ◽  
Single Molecule ◽  
Random Mutagenesis ◽  
Nicotinic Acetylcholine

scholarly journals A Wi-Fi live streaming Centrifuge Force Microscope for benchtop single-molecule experiments

2020 ◽  
Author(s):  
Jibin Abraham Punnoose ◽  
Andrew Hayden ◽  
Lifeng Zhou ◽  
Ken Halvorsen
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Base Pair ◽  
Ease Of Use ◽  
Live Streaming ◽  
Dna Duplexes ◽  
Kinetic Measurements ◽  
Dissociation Kinetic ◽  
The Difference ◽  
Motor Operation

AbstractThe ability to apply controlled forces to individual molecules has been revolutionary in shaping our understanding of biophysics in areas as diverse as dynamic bond strength, biological motor operation, and DNA replication. However, the methodology to perform single-molecule experiments has been and remains relatively inaccessible due to cost and complexity. In 2010, we introduced the Centrifuge Force Microscope (CFM) as a new platform for accessible and high-throughput single-molecule experimentation. The CFM consists of a rotating microscope where prescribed centrifugal forces can be applied to microsphere-tethered biomolecules. In this work, we develop and demonstrate a next-generation Wi-Fi CFM that offers unprecedented ease of use and flexibility in design. The modular CFM unit fits within a standard benchtop centrifuge and connects by Wi-Fi to a external computer for live control and streaming at near gigabit speeds. The use of commercial wireless hardware allows for flexibility in programming and provides a streamlined upgrade path as Wi-Fi technology improves. To facilitate ease of use, detailed build and setup instructions are provided, as well as LabVIEW™ based control software and MATLAB® based analysis software. We demonstrate the analysis of force-dependent dissociation of short DNA duplexes of 7, 8, and 9 bp using the instrument. We showcase the sensitivity of the approach by resolving distinct dissociation kinetic rates for a 7 bp duplex where one G-C base pair is mutated to an A-T base pair.SignificanceThe ability to apply mechanical forces to individual molecules has provided unprecedented insight into many areas of biology. Centrifugal force provides a way to increase the throughput and to decrease the cost and complexity of single-molecule experiments compared to other approaches. In this work, we develop and demonstrate a new user-friendly Centrifuge Force Microscope (CFM) that enables live-streaming of high-throughput single-molecule experiments in a benchtop centrifuge. We achieved near gigabit bandwidth with standard Wi-Fi components, and we provide detailed design instructions and software to facilitate use by other labs. We demonstrate the instrument for sensitive kinetic measurements that are capable of resolving the difference between two DNA duplexes that differ by a single G-C to A-T substitution.

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