scholarly journals Supercoiling DNA optically

2019 ◽  
Vol 116 (52) ◽  
pp. 26534-26539
Author(s):  
Graeme A. King ◽  
Federica Burla ◽  
Erwin J. G. Peterman ◽  
Gijs J. L. Wuite
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Dna Supercoiling ◽  
Biological Interactions ◽  
Mitochondrial Transcription ◽  
Supercoiled Dna ◽  
Single Molecule Level ◽  
Mitochondrial Transcription Factor ◽  
Wide Range

Cellular DNA is regularly subject to torsional stress during genomic processes, such as transcription and replication, resulting in a range of supercoiled DNA structures. For this reason, methods to prepare and study supercoiled DNA at the single-molecule level are widely used, including magnetic, angular-optical, micropipette, and magneto-optical tweezers. However, it is currently challenging to combine DNA supercoiling control with spatial manipulation and fluorescence microscopy. This limits the ability to study complex and dynamic interactions of supercoiled DNA. Here we present a single-molecule assay that can rapidly and controllably generate negatively supercoiled DNA using a standard dual-trap optical tweezers instrument. This method, termed Optical DNA Supercoiling (ODS), uniquely combines the ability to study supercoiled DNA using force spectroscopy, fluorescence imaging of the whole DNA, and rapid buffer exchange. The technique can be used to generate a wide range of supercoiled states, with between <5 and 70% lower helical twist than nonsupercoiled DNA. Highlighting the versatility of ODS, we reveal previously unobserved effects of ionic strength and sequence on the structural state of underwound DNA. Next, we demonstrate that ODS can be used to directly visualize and quantify protein dynamics on supercoiled DNA. We show that the diffusion of the mitochondrial transcription factor TFAM can be significantly hindered by local regions of underwound DNA. This finding suggests a mechanism by which supercoiling could regulate mitochondrial transcription in vivo. Taken together, we propose that ODS represents a powerful method to study both the biophysical properties and biological interactions of negatively supercoiled DNA.

scholarly journals Supercoiling DNA optically

2019 ◽  
Author(s):  
Graeme A. King ◽  
Federica Burla ◽  
Erwin J. G. Peterman ◽  
Gijs J.L. Wuite
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Torque Control ◽  
Biological Interactions ◽  
Supercoiled Dna ◽  
Dna Structures ◽  
Single Molecule Level ◽  
Open Questions ◽  
Cellular Dna ◽  
Transcription And Replication

AbstractCellular DNA is regularly subject to torsional stress during genomic processes, such as transcription and replication, resulting in a range of supercoiled DNA structures.1,2,3 For this reason, methods to prepare and study supercoiled DNA at the single-molecule level are widely used, including magnetic,4,5,6 angular-optical,7,8,9 micro-pipette,10 and magneto-optical tweezers.11 However, in order to address many open questions, there is a growing need for new techniques that can combine rapid torque control with both spatial manipulation and fluorescence microscopy. Here we present a single-molecule assay that can rapidly and controllably generate negatively supercoiled DNA using a standard dual-trap optical tweezers instrument. Supercoiled DNA formed in this way is amenable to fast buffer exchange, and can be interrogated with both force spectroscopy and fluorescence imaging of the whole DNA. We establish this method as a powerful platform to study both the biophysical properties and biological interactions of negatively supercoiled DNA.

scholarly journals DNA fluctuations reveal the size and dynamics of topological domains

2021 ◽  
Author(s):  
Willem Vanderlinden ◽  
Enrico Skoruppa ◽  
Pauline J. Kolbeck ◽  
Enrico Carlon ◽  
Jan Lipfert
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Dna Supercoiling ◽  
Magnetic Tweezers ◽  
Torsional Stiffness ◽  
Molecular Architecture ◽  
Supercoiled Dna ◽  
Topological Domains ◽  
Single Molecule Level ◽  
Processing Enzymes

DNA supercoiling is a key regulatory mechanism that orchestrates DNA readout, recombination, and genome maintenance. DNA-binding proteins often mediate these processes by bringing two distant DNA sites together, thereby inducing (transient) topological domains. In order to understand the dynamics and molecular architecture of protein induced topological domains in DNA, quantitative and time-resolved approaches are required. Here we present a methodology to determine the size and dynamics of topological domains in supercoiled DNA in real-time and at the single molecule level. Our approach is based on quantifying the extension fluctuations -in addition to the mean extension- of supercoiled DNA in magnetic tweezers. Using a combination of high-speed magnetic tweezers experiments, Monte Carlo simulations, and analytical theory, we map out the dependence of DNA extension fluctuations as a function of supercoiling density and external force. We find that in the plectonemic regime the extension variance increases linearly with increasing supercoiling density and show how this enables us to determine the formation and size of topological domains. In addition, we demonstrate how transient (partial) dissociation of DNA bridging proteins results in dynamic sampling of different topological states, which allows us to deduce the torsional stiffness of the plectonemic state and the kinetics of protein-plectoneme interactions. We expect our approach to enable quantification of the dynamics and reaction pathways of DNA processing enzymes and motor proteins, in the context of physiologically relevant forces and supercoiling densities.

scholarly journals Mitochondrial transcription factor A in RORγt+ lymphocytes regulate small intestine homeostasis and metabolism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zheng Fu ◽  
Joseph W. Dean ◽  
Lifeng Xiong ◽  
Michael W. Dougherty ◽  
Kristen N. Oliff ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Small Intestine ◽  
Th17 Cells ◽  
Tissue Remodeling ◽  
Mitochondrial Transcription ◽  
Mitochondrial Transcription Factor ◽  
Tuft Cell ◽  
Mitochondrial Transcription Factor A

AbstractRORγt+ lymphocytes, including interleukin 17 (IL-17)-producing gamma delta T (γδT17) cells, T helper 17 (Th17) cells, and group 3 innate lymphoid cells (ILC3s), are important immune regulators. Compared to Th17 cells and ILC3s, γδT17 cell metabolism and its role in tissue homeostasis remains poorly understood. Here, we report that the tissue milieu shapes splenic and intestinal γδT17 cell gene signatures. Conditional deletion of mitochondrial transcription factor A (Tfam) in RORγt+ lymphocytes significantly affects systemic γδT17 cell maintenance and reduces ILC3s without affecting Th17 cells in the gut. In vivo deletion of Tfam in RORγt+ lymphocytes, especially in γδT17 cells, results in small intestine tissue remodeling and increases small intestine length by enhancing the type 2 immune responses in mice. Moreover, these mice show dysregulation of the small intestine transcriptome and metabolism with less body weight but enhanced anti-helminth immunity. IL-22, a cytokine produced by RORγt+ lymphocytes inhibits IL-13-induced tuft cell differentiation in vitro, and suppresses the tuft cell-type 2 immune circuit and small intestine lengthening in vivo, highlighting its key role in gut tissue remodeling.

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.

Quantitative single molecule FRET efficiencies using TIRF microscopy

2015 ◽  
Vol 184 ◽  
pp. 131-142 ◽  
Author(s):  
Lasse L. Hildebrandt ◽  
Søren Preus ◽  
Victoria Birkedal
Keyword(s):  
Single Molecule ◽  
Structural Information ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Complexes ◽  
Distance Information ◽  
Single Molecule Level ◽  
Single Molecule Fret ◽  
Wide Range ◽  
Intermolecular Distances

Förster resonance energy transfer (FRET) microscopy at the single molecule level has the potential to yield information on intra and intermolecular distances within the 2–10 nm range of molecules or molecular complexes that undergo frequent conformation changes. A pre-requirement for obtaining accurate distance information is to determine quantitative instrument independent FRET efficiency values. Here, we applied and evaluated a procedure to determine quantitative FRET efficiencies directly from individual fluorescence time traces of surface immobilized DNA molecules without the need for external calibrants. To probe the robustness of the approach over a wide range of FRET efficiencies we used a set of doubly labelled double stranded DNA samples, where the acceptor position was varied systematically. Interestingly, we found that fluorescence contributions arising from direct acceptor excitation following donor excitation are intrinsically taken into account in these conditions as other correction factors can compensate for inaccurate values of these parameters. We give here guidelines, that can be used through tools within the iSMS software (http://www.isms.au.dk), for determining quantitative FRET and assess uncertainties linked with the procedure. Our results provide insights into the experimental parameters governing quantitative FRET determination, which is essential for obtaining accurate structural information from a wide range of biomolecules.

scholarly journals Bio-Molecular Applications of Recent Developments in Optical Tweezers

Biomolecules ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 23 ◽  
Author(s):  
Dhawal Choudhary ◽  
Alessandro Mossa ◽  
Milind Jadhav ◽  
Ciro Cecconi
Keyword(s):  
Photonic Crystal ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Continuous Wave ◽  
Imaging Techniques ◽  
Resolving Power ◽  
Optical Traps ◽  
Laser Source ◽  
One Dimensional

In the past three decades, the ability to optically manipulate biomolecules has spurred a new era of medical and biophysical research. Optical tweezers (OT) have enabled experimenters to trap, sort, and probe cells, as well as discern the structural dynamics of proteins and nucleic acids at single molecule level. The steady improvement in OT’s resolving power has progressively pushed the envelope of their applications; there are, however, some inherent limitations that are prompting researchers to look for alternatives to the conventional techniques. To begin with, OT are restricted by their one-dimensional approach, which makes it difficult to conjure an exhaustive three-dimensional picture of biological systems. The high-intensity trapping laser can damage biological samples, a fact that restricts the feasibility of in vivo applications. Finally, direct manipulation of biological matter at nanometer scale remains a significant challenge for conventional OT. A significant amount of literature has been dedicated in the last 10 years to address the aforementioned shortcomings. Innovations in laser technology and advances in various other spheres of applied physics have been capitalized upon to evolve the next generation OT systems. In this review, we elucidate a few of these developments, with particular focus on their biological applications. The manipulation of nanoscopic objects has been achieved by means of plasmonic optical tweezers (POT), which utilize localized surface plasmons to generate optical traps with enhanced trapping potential, and photonic crystal optical tweezers (PhC OT), which attain the same goal by employing different photonic crystal geometries. Femtosecond optical tweezers (fs OT), constructed by replacing the continuous wave (cw) laser source with a femtosecond laser, promise to greatly reduce the damage to living samples. Finally, one way to transcend the one-dimensional nature of the data gained by OT is to couple them to the other large family of single molecule tools, i.e., fluorescence-based imaging techniques. We discuss the distinct advantages of the aforementioned techniques as well as the alternative experimental perspective they provide in comparison to conventional OT.

Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase

2020 ◽  
Author(s):  
Keith J Mickolajczyk ◽  
Patrick M M Shelton ◽  
Michael Grasso ◽  
Xiaocong Cao ◽  
Sara R Warrington ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Helicase Activity ◽  
Structural Protein ◽  
Rna Dependent Rna Polymerase ◽  
Velocity Increase ◽  
Single Molecule Level ◽  
On Demand ◽  
Dependent Behavior ◽  
Stimulation Of

The superfamily-1 helicase non-structural protein 13 (nsp13) is required for SARS-CoV-2 replication, making it an important antiviral therapeutic target. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the dsRNA, suggesting a passive unwinding mechanism. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be potently activated by picoNewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, drawing stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.

scholarly journals Broken force dispersal network in tip-links by the mutations at the Ca2+-binding residues induces hearing-loss

2019 ◽  
Vol 476 (16) ◽  
pp. 2411-2425 ◽  
Author(s):  
Jagadish P. Hazra ◽  
Amin Sagar ◽  
Nisha Arora ◽  
Debadutta Deb ◽  
Simerpreet Kaur ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Inner Ear ◽  
Single Molecule ◽  
Force Sensor ◽  
Force Sensors ◽  
Single Molecule Level ◽  
Conformational Variability ◽  
Atomic Force ◽  
Wide Range ◽  
And Function

Abstract Tip-link as force-sensor in hearing conveys the mechanical force originating from sound to ion-channels while maintaining the integrity of the entire sensory assembly in the inner ear. This delicate balance between structure and function of tip-links is regulated by Ca2+-ions present in endolymph. Mutations at the Ca2+-binding sites of tip-links often lead to congenital deafness, sometimes syndromic defects impairing vision along with hearing. Although such mutations are already identified, it is still not clear how the mutants alter the structure-function properties of the force-sensors associated with diseases. With an aim to decipher the differences in force-conveying properties of the force-sensors in molecular details, we identified the conformational variability of mutant and wild-type tip-links at the single-molecule level using FRET at the endolymphatic Ca2+ concentrations and subsequently measured the force-responsive behavior using single-molecule force spectroscopy with an Atomic Force Microscope (AFM). AFM allowed us to mimic the high and wide range of force ramps (103–106 pN s−1) as experienced in the inner ear. We performed in silico network analysis to learn that alterations in the conformations of the mutants interrupt the natural force-propagation paths through the sensors and make the mutant tip-links vulnerable to input forces from sound stimuli. We also demonstrated that a Ca2+ rich environment can restore the force-response of the mutant tip-links which may eventually facilitate the designing of better therapeutic strategies to the hearing loss.

scholarly journals Profound Nanoscale Structural and Biomechanical Changes in DNA Helix upon Treatment with Anthracycline Drugs

2020 ◽  
Vol 21 (11) ◽  
pp. 4142
Author(s):  
Aleksandra Kaczorowska ◽  
Weronika Lamperska ◽  
Kaja Frączkowska ◽  
Jan Masajada ◽  
Sławomir Drobczyński ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Regulatory Elements ◽  
Dna Molecules ◽  
Anthracycline Antibiotics ◽  
Single Molecule Level ◽  
Force Microscopy ◽  
Atomic Force ◽  
Microscopy Analysis ◽  
Dna Regulatory Elements

In our study, we describe the outcomes of the intercalation of different anthracycline antibiotics in double-stranded DNA at the nanoscale and single molecule level. Atomic force microscopy analysis revealed that intercalation results in significant elongation and thinning of dsDNA molecules. Additionally, using optical tweezers, we have shown that intercalation decreases the stiffness of DNA molecules, that results in greater susceptibility of dsDNA to break. Using DNA molecules with different GC/AT ratios, we checked whether anthracycline antibiotics show preference for GC-rich or AT-rich DNA fragments. We found that elongation, decrease in height and decrease in stiffness of dsDNA molecules was highest in GC-rich dsDNA, suggesting the preference of anthracycline antibiotics for GC pairs and GC-rich regions of DNA. This is important because such regions of genomes are enriched in DNA regulatory elements. By using three different anthracycline antibiotics, namely doxorubicin (DOX), epirubicin (EPI) and daunorubicin (DAU), we could compare their detrimental effects on DNA. Despite their analogical structure, anthracyclines differ in their effects on DNA molecules and GC-rich region preference. DOX had the strongest overall effect on the DNA topology, causing the largest elongation and decrease in height. On the other hand, EPI has the lowest preference for GC-rich dsDNA. Moreover, we demonstrated that the nanoscale perturbations in dsDNA topology are reflected by changes in the microscale properties of the cell, as even short exposition to doxorubicin resulted in an increase in nuclei stiffness, which can be due to aberration of the chromatin organization, upon intercalation of doxorubicin molecules.

Sign in / Sign up

Export Citation Format

Share Document