scholarly journals Multiplex flow magnetic tweezers reveal rare enzymatic events with single molecule precision

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Rohit Agarwal ◽  
Karl E. Duderstadt
Keyword(s):  
Single Molecule ◽  
Rare Events ◽  
Dynamic Properties ◽  
Magnetic Tweezers ◽  
Drug Induced ◽  
Single Molecule Level ◽  
Dna Break ◽  
Reaction Cycles ◽  
Enzymatic Pathways ◽  
Routine Imaging

Abstract The application of forces and torques on the single molecule level has transformed our understanding of the dynamic properties of biomolecules, but rare intermediates have remained difficult to characterize due to limited throughput. Here, we describe a method that provides a 100-fold improvement in the throughput of force spectroscopy measurements with topological control, which enables routine imaging of 50,000 single molecules and a 100 million reaction cycles in parallel. This improvement enables detection of rare events in the life cycle of the cell. As a demonstration, we characterize the supercoiling dynamics and drug-induced DNA break intermediates of topoisomerases. To rapidly quantify distinct classes of dynamic behaviors and rare events, we developed a software platform with an automated feature classification pipeline. The method and software can be readily adapted for studies of a broad range of complex, multistep enzymatic pathways in which rare intermediates have escaped classification due to limited throughput.

Magnetic tweezers: a sensitive tool to study DNA and chromatin at the single-molecule level

2003 ◽  
Vol 81 (3) ◽  
pp. 151-159 ◽  
Author(s):  
Jordanka Zlatanova ◽  
Sanford H Leuba
Keyword(s):  
Single Molecule ◽  
Magnetic Tweezers ◽  
Biological Macromolecules ◽  
High Definition ◽  
Single Molecule Level ◽  
Applied Tension ◽  
Sensitive Tool ◽  
Different Types ◽  
Distinct Features ◽  
Insight Into

The advent of single-molecule biology has allowed unprecedented insight into the dynamic behavior of biological macromolecules and their complexes. Unexpected properties, masked by the asynchronous behavior of myriads of molecules in bulk experiments, can be revealed; equally importantly, individual members of a molecular population often exhibit distinct features in their properties. Finally, the single-molecule approaches allow us to study the behavior of biological macromolecules under applied tension or torsion; understanding the mechanical properties of these molecules helps us understand how they function in the cell. In this review, we summarize the application of magnetic tweezers (MT) to the study of DNA behavior at the single-molecule level. MT can be conveniently used to stretch DNA and introduce controlled levels of superhelicity into the molecule and to follow to a high definition the action of different types of topoisomerases. Its potential for chromatin studies is also enormous, and we will briefly present our first chromatin results.Key words: single-molecules, chromatin, topoisomerases, magnetic tweezers, force.

scholarly journals A Study of p53 Action on DNA at the Single Molecule Level

2021 ◽  
Author(s):  
Kiyoto Kamagata
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Dynamic Properties ◽  
Diffusion Mechanism ◽  
Target Search ◽  
Single Molecule Level ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Biophysical Study ◽  
Model Protein

The transcription factor p53 searches for and binds to target sequences within long genomic DNA, to regulate downstream gene expression. p53 possesses multiple disordered and DNA-binding domains, which are frequently observed in DNA-binding proteins. Owing to these properties, p53 is used as a model protein for target search studies. It counters cell stress by utilizing a facilitated diffusion mechanism that combines 3D diffusion in solution, 1D sliding along DNA, hopping/jumping along DNA, and intersegmental transfer between two DNAs. Single-molecule fluorescence microscopy has been used to characterize individual motions of p53 in detail. In addition, a biophysical study has revealed that p53 forms liquid-like droplets involving the functional switch. In this chapter, the target search and regulation of p53 are discussed in terms of dynamic properties.

scholarly journals A flow extension tethered particle motion assay for single-molecule proteolysis

2019 ◽  
Author(s):  
Andrew A. Drabek ◽  
Joseph J. Loparo ◽  
Stephen C. Blacklow
Keyword(s):  
Single Molecule ◽  
Magnetic Force ◽  
Magnetic Tweezers ◽  
Signaling Proteins ◽  
Mechanical Tension ◽  
Tobacco Etch Virus Protease ◽  
Single Molecule Level ◽  
Wide Range ◽  
Tethered Particle Motion

AbstractRegulated proteolysis of signaling proteins under mechanical tension enables cells to communicate with their environment in a variety of developmental and physiologic contexts. The role of force in inducing proteolytic sensitivity has been explored using magnetic tweezers at the single-molecule level with bead-tethered assays, but such efforts have been limited by challenges in ensuring that beads are not restrained by multiple tethers. Here, we describe a multiplexed assay for single-molecule proteolysis that overcomes the multiple-tether problem using a flow extension (FLEX) strategy on a microscope equipped with magnetic tweezers. Particle tracking and computational sorting of flow-induced displacements allows assignment of tethered substrates into singly-captured and multiply-tethered bins, with the fraction of fully mobile, single-tethered substrates depending inversely on the concentration of substrate loaded on the coverslip. Computational exclusion of multiply-tethered beads enables robust assessment of on-target proteolysis by the highly specific tobacco etch virus protease and the more promiscuous metalloprotease ADAM17. This method should be generally applicable to a wide range of proteases and readily extensible to robust evaluation of proteolytic sensitivity as a function of applied magnetic force.

Single-Molecule Level Rare Events Revealed by Dynamic Surface-Enhanced Raman Spectroscopy

2020 ◽  
Vol 92 (24) ◽  
pp. 15806-15810
Author(s):  
Cheng Zong ◽  
Chan-juan Chen ◽  
Xiang Wang ◽  
Pei Hu ◽  
Guo-kun Liu ◽  
...  
Keyword(s):  
Raman Spectroscopy ◽  
Single Molecule ◽  
Rare Events ◽  
Surface Enhanced Raman Spectroscopy ◽  
Dynamic Surface ◽  
Single Molecule Level ◽  
Surface Enhanced ◽  
Surface Enhanced Raman

Studies of DNA-Replication at the Single Molecule Level Using Magnetic Tweezers

2010 ◽  
pp. 89-122
Author(s):  
Maria Manosas ◽  
Timothée Lionnet ◽  
Élise Praly ◽  
Ding Fangyuan ◽  
Jean-François Allemand ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Magnetic Tweezers ◽  
Single Molecule Level

Magnetic tweezers for the mechanical research of DNA at the single molecule level

2017 ◽  
Vol 9 (39) ◽  
pp. 5720-5730 ◽  
Author(s):  
Qi Xin ◽  
Peng Li ◽  
Yuning He ◽  
Cuiping Shi ◽  
Yiqun Qiao ◽  
...  
Keyword(s):  
Single Molecule ◽  
Magnetic Tweezers ◽  
Single Molecule Level

This review summarizes the application of magnetic tweezers for the mechanical research of DNA at the single molecule level.

scholarly journals Identical Sequences, Different Behaviors: Protein Diversity Captured at the Single-Molecule Level

2021 ◽  
Author(s):  
Rafael Tapia-Rojo ◽  
Alvaro Alonso-Caballero ◽  
Carmen L. Badilla ◽  
Julio M. Fernandez
Keyword(s):  
Single Molecule ◽  
Magnetic Tweezers ◽  
Complete Characterization ◽  
Force Response ◽  
Folding Kinetics ◽  
Single Molecule Level ◽  
Biochemical Assays ◽  
Folding Dynamics ◽  
Instrumental Development

AbstractThe classical “one sequence, one structure, one function” paradigm has shaped much of our intuition of how proteins work inside the cell. Partially due to the insight provided by bulk biochemical assays, individual biomolecules are assumed to behave as identical entities, and their characterization relies on ensemble averages that flatten any conformational diversity into a unique phenotype. While the emergence of single-molecule techniques opened the gates to interrogating individual molecules, technical shortcomings typically limit the duration of these measurements to a few minutes, which prevents to completely characterize a protein individual and, hence, to capture the heterogeneity among molecular populations. Here, we introduce a magnetic tweezers design, which showcases enhanced stability and resolution that allows us to measure the folding dynamics of a single protein during several uninterrupted days with a high temporal and spatial resolution. Thanks to this instrumental development, we do a complete characterization of two proteins with a very different force-response: the talin R3IVVI domain and protein L. Days-long recordings on the same single molecule accumulate several thousands of folding transitions sampled with sub-ms resolution, which allows us to reconstruct their free energy landscapes and describe how they evolve with force. By mapping the nanomechanical identity of many different protein individuals, we directly capture their molecular diversity as a quantifiable dispersion on their force response and folding kinetics. Our instrumental development offers a new tool for profiling individual molecules, opening the gates to the characterization of biomolecular heterogeneity.

scholarly journals Histone H1 compacts DNA under force and during chromatin assembly

2012 ◽  
Vol 23 (24) ◽  
pp. 4864-4871 ◽  
Author(s):  
Botao Xiao ◽  
Benjamin S. Freedman ◽  
Kelly E. Miller ◽  
Rebecca Heald ◽  
John F. Marko
Keyword(s):  
Single Molecule ◽  
Physiological Role ◽  
Histone H1 ◽  
Magnetic Tweezers ◽  
Chromatin Assembly ◽  
Compaction Rate ◽  
Single Molecule Level ◽  
Single Dna Molecules ◽  
Naked Dna ◽  
Egg Extracts

Histone H1 binds to linker DNA between nucleosomes, but the dynamics and biological ramifications of this interaction remain poorly understood. We performed single-molecule experiments using magnetic tweezers to determine the effects of H1 on naked DNA in buffer or during chromatin assembly in Xenopus egg extracts. In buffer, nanomolar concentrations of H1 induce bending and looping of naked DNA at stretching forces below 0.6 pN, effects that can be reversed with 2.7-pN force or in 200 mM monovalent salt concentrations. Consecutive tens-of-nanometer bending events suggest that H1 binds to naked DNA in buffer at high stoichiometries. In egg extracts, single DNA molecules assemble into nucleosomes and undergo rapid compaction. Histone H1 at endogenous physiological concentrations increases the DNA compaction rate during chromatin assembly under 2-pN force and decreases it during disassembly under 5-pN force. In egg cytoplasm, histone H1 protects sperm nuclei undergoing genome-wide decondensation and chromatin assembly from becoming abnormally stretched or fragmented due to astral microtubule pulling forces. These results reveal functional ramifications of H1 binding to DNA at the single-molecule level and suggest an important physiological role for H1 in compacting DNA under force and during chromatin assembly.

Sign in / Sign up

Export Citation Format

Share Document