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

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.

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Tracking On-Surface Chemistry with Atomic Precision

Synlett ◽

10.1055/s-0036-1590867 ◽

2017 ◽

Vol 28 (19) ◽

pp. 2509-2516 ◽

Cited By ~ 5

Author(s):

Peter Jacobse ◽

Marc-Etienne Moret ◽

Robertus Klein Gebbink ◽

Ingmar Swart

Keyword(s):

Atomic Force Microscopy ◽

Single Molecule ◽

Graphene Nanoribbons ◽

Single Molecule Level ◽

Force Microscopy ◽

The Past ◽

Atomic Force ◽

Atomic Precision ◽

Different Types ◽

Insight Into

The field of on-surface synthesis has seen a tremendous development in the past decade as an exciting new methodology towards atomically well-defined nanostructures. A strong driving force in this respect is its inherent compatibility with scanning probe techniques, which allows one to ‘view’ the reactants and products at the single-molecule level. In this article, we review the ability of noncontact atomic force microscopy to study on-surface chemical reactions with atomic precision. We highlight recent advances in using noncontact atomic force microscopy to obtain mechanistic insight into reactions and focus on the recently elaborated mechanisms in the formation of different types of graphene nanoribbons.

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Constructing a Magnetic Tweezers to Monitor RNA Translocation at the Single-Molecule Level

Methods in Molecular Biology - RNA Remodeling Proteins ◽

10.1007/978-1-4939-2214-7_16 ◽

2014 ◽

pp. 257-273

Author(s):

Desiree Salas ◽

Veronika Gocheva ◽

Marcelo Nöllmann

Keyword(s):

Single Molecule ◽

Magnetic Tweezers ◽

Single Molecule Level

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Dynamics of Tpm1.8 domains on actin filaments with single molecule resolution

10.1101/2020.06.15.152033 ◽

2020 ◽

Cited By ~ 1

Author(s):

Ilina Bareja ◽

Hugo Wioland ◽

Miro Janco ◽

Philip R. Nicovich ◽

Antoine Jégou ◽

...

Keyword(s):

Actin Filament ◽

Single Molecule ◽

Actin Filaments ◽

High Probability ◽

Actin Binding ◽

Single Molecule Level ◽

Filament Dynamics ◽

Kinetics Of ◽

Insight Into

ABSTRACTTropomyosins regulate dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of fluorescently labelled tropomyosin isoform Tpm1.8 to unlabelled actin filaments in real time. This approach in conjunction with mathematical modeling enabled us to quantify the nucleation, assembly and disassembly kinetics of Tpm1.8 on single filaments and at the single molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilised by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented towards the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

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Annealing synchronizes the 70S ribosome into a minimum-energy conformation

10.1101/2021.06.25.447849 ◽

2021 ◽

Author(s):

Xiaofeng Chu ◽

Xin Su ◽

Mingdong Liu ◽

Li Li ◽

Tianhao Li ◽

...

Keyword(s):

Free Energy ◽

Single Molecule ◽

Energy Landscape ◽

Minimum Energy ◽

Boltzmann Distribution ◽

Minimum Free Energy ◽

Biological Macromolecules ◽

Single Molecule Level ◽

30S Subunit ◽

70S Ribosome

Researchers commonly anneal metals, alloys, and semiconductors to repair defects and improve microstructures via recrystallization. Theoretical studies indicate simulated annealing on biological macromolecules helps predict the final structures with minimum free energy. Experimental validation of this homogenizing effect and further exploration of its applications are fascinating scientific questions that remain elusive. Here, we chose the apo-state 70S ribosome from Escherichia coli as a model, wherein the 30S subunit undergoes a thermally driven inter-subunit rotation and exhibits substantial structural flexibility as well as distinct free energy. We experimentally demonstrate that annealing at a fast cooling rate enhances the 70S ribosome homogeneity and improves local resolution on the 30S subunit. After annealing, the 70S ribosome is in a nonrotated state with respect to corresponding intermediate structures in unannealed or heated ribosomes, and exhibits a minimum energy in the free energy landscape. One can readily crystallize these minimum-energy ribosomes, which have great potential for synchronizing proteins on a single-molecule level. Our experimental results are consistent with theoretical analysis on the temperature-dependent Boltzmann distribution, and offer a facile yet robust approach to enhance protein stability, which is ideal for high-resolution cryogenic electron microscopy. Beyond structure determination, annealing can be extended to study protein folding and explore conformational and energy landscape.

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

Nature Communications ◽

10.1038/s41467-020-18456-y ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 2

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.

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Electron transfer behaviour of biological macromolecules towards the single-molecule level

Journal of Physics Condensed Matter ◽

10.1088/0953-8984/15/18/318 ◽

2003 ◽

Vol 15 (18) ◽

pp. S1873-S1890 ◽

Cited By ~ 16

Author(s):

Jingdong Zhang ◽

Mikala Grubb ◽

Allan G Hansen ◽

Alexander M Kuznetsov ◽

Anja Boisen ◽

...

Keyword(s):

Electron Transfer ◽

Single Molecule ◽

Biological Macromolecules ◽

Single Molecule Level

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Studies of DNA-Protein Interactions at the Single Molecule Level with Magnetic Tweezers

Controlled Nanoscale Motion - Lecture Notes in Physics ◽

10.1007/3-540-49522-3_6 ◽

2007 ◽

pp. 123-140 ◽

Cited By ~ 1

Author(s):

J.-F. Allemand ◽

D. Bensimon ◽

G. Charvin ◽

V. Croquette ◽

G. Lia ◽

...

Keyword(s):

Single Molecule ◽

Protein Interactions ◽

Magnetic Tweezers ◽

Single Molecule Level ◽

Dna Protein Interactions

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

10.1101/528919 ◽

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.

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Insight into helicase mechanism and function revealed through single-molecule approaches

Quarterly Reviews of Biophysics ◽

10.1017/s0033583510000107 ◽

2010 ◽

Vol 43 (2) ◽

pp. 185-217 ◽

Cited By ~ 46

Author(s):

Jaya G. Yodh ◽

Michael Schlierf ◽

Taekjip Ha

Keyword(s):

Single Molecule ◽

Conformational Dynamics ◽

Magnetic Tweezers ◽

Repetitive Behaviors ◽

Helicase Activity ◽

Step Size ◽

Mechanistic Insight ◽

Combination Of Methods ◽

And Function ◽

Insight Into

AbstractHelicases are a class of nucleic acid (NA) motors that catalyze NTP-dependent unwinding of NA duplexes into single strands, a reaction essential to all areas of NA metabolism. In the last decade, single-molecule (sm) technology has proven to be highly useful in revealing mechanistic insight into helicase activity that is not always detectable via ensemble assays. A combination of methods based on fluorescence, optical and magnetic tweezers, and flow-induced DNA stretching has enabled the study of helicase conformational dynamics, force generation, step size, pausing, reversal and repetitive behaviors during translocation and unwinding by helicases working alone and as part of multiprotein complexes. The contributions of these sm investigations to our understanding of helicase mechanism and function will be discussed.

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