scholarly journals Force-dependent persistence length of DNA–intercalator complexes measured in single molecule stretching experiments

Soft Matter ◽  
2015 ◽  
Vol 11 (21) ◽  
pp. 4306-4314 ◽  
Author(s):  
R. F. Bazoni ◽  
C. H. M. Lima ◽  
E. B. Ramos ◽  
M. S. Rocha
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Mechanical Response ◽  
Persistence Length ◽  
Dna Intercalator ◽  
Applied Forces

By using optical tweezers with an adjustable trap stiffness, we have performed systematic single molecule stretching experiments with two types of DNA–intercalator complexes, in order to investigate the effects of the maximum applied forces on the mechanical response of such complexes.

scholarly journals Understanding the paradoxical mechanical response of in-phase A-tracts at different force regimes

2019 ◽  
Author(s):  
Alberto Marin-Gonzalez ◽  
Cesar L. Pastrana ◽  
Rebeca Bocanegra ◽  
Alejandro Martín-González ◽  
J.G. Vilhena ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Dna Sequences ◽  
Mechanical Response ◽  
Magnetic Tweezers ◽  
Crystallographic Structure ◽  
Chain Model ◽  
Microscopy Imaging ◽  
Length Scales

ABSTRACTA-tracts are A:T rich DNA sequences that exhibit unique structural and mechanical properties associated with several functions in vivo. The crystallographic structure of A-tracts has been well characterized. However, their response to forces remains unknown and the variability of their flexibility reported for different length scales has precluded a comprehensive description of the mechanical properties of these molecules. Here, we rationalize the mechanical properties of A-tracts across multiple length scales using a combination of single-molecule experiments and theoretical polymer models applied to DNA sequences present in the C. elegans genome. Atomic Force Microscopy imaging shows that phased A-tracts induce long-range (∼200 nm) bending. Moreover, the enhanced bending originates from an intrinsically bent structure rather than as a consequence of larger flexibility. In support of this, our data were well described with a theoretical model based on the worm-like chain model that includes intrinsic bending. Magnetic tweezers experiments confirm that the observed bent is intrinsic to the sequence and does not rely on particular ionic conditions. Using optical tweezers, we assess the local rigidity of A-tracts at high forces and unravel an unusually stiff character of these sequences, as quantified by their large stretch modulus. Our work rationalizes the complex multiscale flexibility of A-tracts, shedding light on the cryptic character of these sequences.

scholarly journals Single-molecule mechanics of protein-labelled DNA handles

2016 ◽  
Vol 7 ◽  
pp. 138-148 ◽  
Author(s):  
Vivek S Jadhav ◽  
Dorothea Brüggemann ◽  
Florian Wruck ◽  
Martin Hegner
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Mechanical Characteristics ◽  
Molecular Motor ◽  
Optical Force ◽  
Depth Information ◽  
Physiological Conditions ◽  
Modified Dna ◽  
Applied Forces ◽  
Surface Modified

DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA–protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG)-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp) were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG) beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD) imaging control experiments revealed that quantum dot–streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein–DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular recognition in time-critical molecular motor studies.

scholarly journals Hydrophobic catalysis and a potential biological role of DNA unstacking induced by environment effects

2019 ◽  
Vol 116 (35) ◽  
pp. 17169-17174 ◽  
Author(s):  
Bobo Feng ◽  
Robert P. Sosa ◽  
Anna K. F. Mårtensson ◽  
Kai Jiang ◽  
Alex Tong ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Hydrophobic Interactions ◽  
Persistence Length ◽  
Poly Ethylene Glycol ◽  
Glycol Ethers ◽  
Base Stacking ◽  
Poly Ethylene ◽  
Dna Double Helix

Hydrophobic base stacking is a major contributor to DNA double-helix stability. We report the discovery of specific unstacking effects in certain semihydrophobic environments. Water-miscible ethylene glycol ethers are found to modify structure, dynamics, and reactivity of DNA by mechanisms possibly related to a biologically relevant hydrophobic catalysis. Spectroscopic data and optical tweezers experiments show that base-stacking energies are reduced while base-pair hydrogen bonds are strengthened. We propose that a modulated chemical potential of water can promote “longitudinal breathing” and the formation of unstacked holes while base unpairing is suppressed. Flow linear dichroism in 20% diglyme indicates a 20 to 30% decrease in persistence length of DNA, supported by an increased flexibility in single-molecule nanochannel experiments in poly(ethylene glycol). A limited (3 to 6%) hyperchromicity but unaffected circular dichroism is consistent with transient unstacking events while maintaining an overall average B-DNA conformation. Further information about unstacking dynamics is obtained from the binding kinetics of large thread-intercalating ruthenium complexes, indicating that the hydrophobic effect provides a 10 to 100 times increased DNA unstacking frequency and an “open hole” population on the order of 10−2 compared to 10−4 in normal aqueous solution. Spontaneous DNA strand exchange catalyzed by poly(ethylene glycol) makes us propose that hydrophobic residues in the L2 loop of recombination enzymes RecA and Rad51 may assist gene recombination via modulation of water activity near the DNA helix by hydrophobic interactions, in the manner described here. We speculate that such hydrophobic interactions may have catalytic roles also in other biological contexts, such as in polymerases.

scholarly journals Understanding the paradoxical mechanical response of in-phase A-tracts at different force regimes

2020 ◽  
Vol 48 (9) ◽  
pp. 5024-5036
Author(s):  
Alberto Marin-Gonzalez ◽  
Cesar L Pastrana ◽  
Rebeca Bocanegra ◽  
Alejandro Martín-González ◽  
J G Vilhena ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Dna Sequences ◽  
Mechanical Response ◽  
Magnetic Tweezers ◽  
Crystallographic Structure ◽  
Chain Model ◽  
Microscopy Imaging ◽  
Wide Range

Abstract A-tracts are A:T rich DNA sequences that exhibit unique structural and mechanical properties associated with several functions in vivo. The crystallographic structure of A-tracts has been well characterized. However, the mechanical properties of these sequences is controversial and their response to force remains unexplored. Here, we rationalize the mechanical properties of in-phase A-tracts present in the Caenorhabditis elegans genome over a wide range of external forces, using single-molecule experiments and theoretical polymer models. Atomic Force Microscopy imaging shows that A-tracts induce long-range (∼200 nm) bending, which originates from an intrinsically bent structure rather than from larger bending flexibility. These data are well described with a theoretical model based on the worm-like chain model that includes intrinsic bending. Magnetic tweezers experiments show that the mechanical response of A-tracts and arbitrary DNA sequences have a similar dependence with monovalent salt supporting that the observed A-tract bend is intrinsic to the sequence. Optical tweezers experiments reveal a high stretch modulus of the A-tract sequences in the enthalpic regime. Our work rationalizes the complex multiscale flexibility of A-tracts, providing a physical basis for the versatile character of these sequences inside the cell.

scholarly journals Optical tweezers in single-molecule biophysics

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Carlos J. Bustamante ◽  
Yann R. Chemla ◽  
Shixin Liu ◽  
Michelle D. Wang
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Single Molecule Biophysics

scholarly journals Anomalous Laterally Stressed Kinetically Trapped DNA Surface Conformations

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Valery V. Prokhorov ◽  
Nikolay A. Barinov ◽  
Kirill A. Prusakov ◽  
Evgeniy V. Dubrovin ◽  
Maxim D. Frank-Kamenetskii ◽  
...  
Keyword(s):  
Electrostatic Interaction ◽  
Mechanical Response ◽  
Persistence Length ◽  
Strong Impact ◽  
List Type ◽  
Surface Charges ◽  
Monomolecular Films ◽  
Chain Approximation ◽  
The Impact ◽  
Positively Charged

Highlights DNA kinking is inevitable for the highly anisotropic 1D–1D electrostatic interaction with the one-dimensionally periodically charged surface. The double helical structure of the DNA kinetically trapped on positively charged monomolecular films comprising the lamellar templates is strongly laterally stressed and extremely perturbed at the nanometer scale. The DNA kinetic trapping is not a smooth 3D—> 2D conformational flattening but is a complex nonlinear in-plane mechanical response (bending, tensile and unzipping) driven by the physics beyond the scope of the applicability of the linear worm-like chain approximation. Abstract Up to now, the DNA molecule adsorbed on a surface was believed to always preserve its native structure. This belief implies a negligible contribution of lateral surface forces during and after DNA adsorption although their impact has never been elucidated. High-resolution atomic force microscopy was used to observe that stiff DNA molecules kinetically trapped on monomolecular films comprising one-dimensional periodically charged lamellar templates as a single layer or as a sublayer are oversaturated by sharp discontinuous kinks and can also be locally melted and supercoiled. We argue that kink/anti-kink pairs are induced by an overcritical lateral bending stress (> 30 pNnm) inevitable for the highly anisotropic 1D-1D electrostatic interaction of DNA and underlying rows of positive surface charges. In addition, the unexpected kink-inducing mechanical instability in the shape of the template-directed DNA confined between the positively charged lamellar sides is observed indicating the strong impact of helicity. The previously reported anomalously low values of the persistence length of the surface-adsorbed DNA are explained by the impact of the surface-induced low-scale bending. The sites of the local melting and supercoiling are convincingly introduced as other lateral stress-induced structural DNA anomalies by establishing a link with DNA high-force mechanics. The results open up the study in the completely unexplored area of the principally anomalous kinetically trapped DNA surface conformations in which the DNA local mechanical response to the surface-induced spatially modulated lateral electrostatic stress is essentially nonlinear. The underlying rich and complex in-plane nonlinear physics acts at the nanoscale beyond the scope of applicability of the worm-like chain approximation.

Molecular Motors: Force and Movement Generated by Single Myosin II Molecules

Physiology ◽  
2002 ◽  
Vol 17 (5) ◽  
pp. 213-218 ◽  
Author(s):  
Caspar Rüegg ◽  
Claudia Veigel ◽  
Justin E. Molloy ◽  
Stephan Schmitz ◽  
John C. Sparrow ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Molecular Motors ◽  
Molecular Motor ◽  
Myosin Ii ◽  
Force Production ◽  
Myosin Isoforms ◽  
Single Molecule Level ◽  
Recent Developments ◽  
Muscle Myosin

Muscle myosin II is an ATP-driven, actin-based molecular motor. Recent developments in optical tweezers technology have made it possible to study movement and force production on the single-molecule level and to find out how different myosin isoforms may have adapted to their specific physiological roles.

scholarly journals Single-molecule force spectroscopy reveals folding steps associated with hormone binding and activation of the glucocorticoid receptor

2018 ◽  
Vol 115 (46) ◽  
pp. 11688-11693 ◽  
Author(s):  
Thomas Suren ◽  
Daniel Rutz ◽  
Patrick Mößmer ◽  
Ulrich Merkel ◽  
Johannes Buchner ◽  
...  
Keyword(s):  
Free Energy ◽  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Mechanical Load ◽  
Force Spectroscopy ◽  
Folding Pathway ◽  
Hormone Binding ◽  
Single Molecule Force Spectroscopy

The glucocorticoid receptor (GR) is a prominent nuclear receptor linked to a variety of diseases and an important drug target. Binding of hormone to its ligand binding domain (GR-LBD) is the key activation step to induce signaling. This process is tightly regulated by the molecular chaperones Hsp70 and Hsp90 in vivo. Despite its importance, little is known about GR-LBD folding, the ligand binding pathway, or the requirement for chaperone regulation. In this study, we have used single-molecule force spectroscopy by optical tweezers to unravel the dynamics of the complete pathway of folding and hormone binding of GR-LBD. We identified a “lid” structure whose opening and closing is tightly coupled to hormone binding. This lid is located at the N terminus without direct contacts to the hormone. Under mechanical load, apo-GR-LBD folds stably and readily without the need of chaperones with a folding free energy of 41 kBT (24 kcal/mol). The folding pathway is largely independent of the presence of hormone. Hormone binds only in the last step and lid closure adds an additional 12 kBT of free energy, drastically increasing the affinity. However, mechanical double-jump experiments reveal that, at zero force, GR-LBD folding is severely hampered by misfolding, slowing it to less than 1·s−1. From the force dependence of the folding rates, we conclude that the misfolding occurs late in the folding pathway. These features are important cornerstones for understanding GR activation and its tight regulation by chaperones.

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