Studying Nucleic Acid–Drug Interactions at the Single-Molecule Level Using Optical Tweezers

2016 ◽  
pp. 154-177
Keyword(s):  
Nucleic Acid ◽  
Drug Interactions ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Single Molecule Level ◽  
Nucleic Acid Drug

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 Targeted binding of nucleocapsid protein transforms the folding landscape of HIV-1 TAR RNA

2015 ◽  
Vol 112 (44) ◽  
pp. 13555-13560 ◽  
Author(s):  
Micah J. McCauley ◽  
Ioulia Rouzina ◽  
Kelly A. Manthei ◽  
Robert J. Gorelick ◽  
Karin Musier-Forsyth ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Transition State ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Reverse Transcription ◽  
Binding Sites ◽  
Specific Binding ◽  
Hybrid Structure ◽  
Tar Rna ◽  
Hiv 1

Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play a key role in the viral life cycle. During reverse transcription, HIV-1 NC facilitates the rearrangement of nucleic acid secondary structure, allowing the transactivation response (TAR) RNA hairpin to be transiently destabilized and annealed to a cDNA hairpin. It is not clear how NC specifically destabilizes TAR RNA but does not strongly destabilize the resulting annealed RNA–DNA hybrid structure, which must be formed for reverse transcription to continue. By combining single-molecule optical tweezers measurements with a quantitative mfold-based model, we characterize the equilibrium TAR stability and unfolding barrier for TAR RNA. Experiments show that adding NC lowers the transition state barrier height while also dramatically shifting the barrier location. Incorporating TAR destabilization by NC into the mfold-based model reveals that a subset of preferential protein binding sites is responsible for the observed changes in the unfolding landscape, including the unusual shift in the transition state. We measure the destabilization induced at these NC binding sites and find that NC preferentially targets TAR RNA by binding to specific sequence contexts that are not present on the final annealed RNA–DNA hybrid structure. Thus, specific binding alters the entire RNA unfolding landscape, resulting in the dramatic destabilization of this specific structure that is required for reverse transcription.

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 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.

PRINCIPLES OF SINGLE-MOLECULE MANIPULATION AND ITS APPLICATION IN BIOLOGICAL PHYSICS

2012 ◽  
Vol 26 (13) ◽  
pp. 1230006 ◽  
Author(s):  
WEI-HUNG CHEN ◽  
JONATHAN D. WILSON ◽  
SITHARA S. WIJERATNE ◽  
SARAH A. SOUTHMAYD ◽  
KUAN-JIUH LIN ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Biological Molecules ◽  
Force Detection ◽  
Biomolecular Systems ◽  
Single Molecule Level ◽  
Detection Techniques ◽  
Atomic Force ◽  
Manipulation System ◽  
Single Molecule Manipulation

Recent advances in nanoscale manipulation and piconewton force detection provide a unique tool for studying the mechanical and thermodynamic properties of biological molecules and complexes at the single-molecule level. Detailed equilibrium and dynamics information on proteins and DNA have been revealed by single-molecule manipulation and force detection techniques. The atomic force microscope (AFM) and optical tweezers have been widely used to quantify the intra- and inter-molecular interactions of many complex biomolecular systems. In this article, we describe the background, analysis, and applications of these novel techniques. Experimental procedures that can serve as a guide for setting up a single-molecule manipulation system using the AFM are also presented.

scholarly journals Nucleic acid-based nanoengineering: novel structures for biomedical applications

2011 ◽  
Vol 1 (5) ◽  
pp. 702-724 ◽  
Author(s):  
Hanying Li ◽  
Thomas H. LaBean ◽  
Kam W. Leong
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Single Molecule ◽  
Biomedical Applications ◽  
Three Dimensional ◽  
Base Pairs ◽  
Single Molecule Level ◽  
Stimulus Responsive ◽  
Bioactive Agents ◽  
Novel Structures

Nanoengineering exploits the interactions of materials at the nanometre scale to create functional nanostructures. It relies on the precise organization of nanomaterials to achieve unique functionality. There are no interactions more elegant than those governing nucleic acids via Watson–Crick base-pairing rules. The infinite combinations of DNA/RNA base pairs and their remarkable molecular recognition capability can give rise to interesting nanostructures that are only limited by our imagination. Over the past years, creative assembly of nucleic acids has fashioned a plethora of two-dimensional and three-dimensional nanostructures with precisely controlled size, shape and spatial functionalization. These nanostructures have been precisely patterned with molecules, proteins and gold nanoparticles for the observation of chemical reactions at the single molecule level, activation of enzymatic cascade and novel modality of photonic detection, respectively. Recently, they have also been engineered to encapsulate and release bioactive agents in a stimulus-responsive manner for therapeutic applications. The future of nucleic acid-based nanoengineering is bright and exciting. In this review, we will discuss the strategies to control the assembly of nucleic acids and highlight the recent efforts to build functional nucleic acid nanodevices for nanomedicine.

scholarly journals Kinetic Characterization of Non-Muscle Myosin IIB Single-Headed Heavy Meromyosin on Single Molecule Level with Optical Tweezers

2010 ◽  
Vol 98 (3) ◽  
pp. 561a
Author(s):  
Attila Nagy ◽  
Yasuharu Takagi ◽  
Earl E. Homsher ◽  
Davin K.T. Hong ◽  
Mihaly Kovacs ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Kinetic Characterization ◽  
Heavy Meromyosin ◽  
Single Molecule Level ◽  
Muscle Myosin

scholarly journals α-Actinin/titin interaction: A dynamic and mechanically stable cluster of bonds in the muscle Z-disk

2017 ◽  
Vol 114 (5) ◽  
pp. 1015-1020 ◽  
Author(s):  
Marco Grison ◽  
Ulrich Merkel ◽  
Julius Kostan ◽  
Kristina Djinović-Carugo ◽  
Matthias Rief
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Kinetic Properties ◽  
Single Molecule Level ◽  
Passive Stretching ◽  
Dual Beam ◽  
Important Interaction ◽  
Cross Linker ◽  
The Individual

Stable anchoring of titin within the muscle Z-disk is essential for preserving muscle integrity during passive stretching. One of the main candidates for anchoring titin in the Z-disk is the actin cross-linker α-actinin. The calmodulin-like domain of α-actinin binds to the Z-repeats of titin. However, the mechanical and kinetic properties of this important interaction are still unknown. Here, we use a dual-beam optical tweezers assay to study the mechanics of this interaction at the single-molecule level. A single interaction of α-actinin and titin turns out to be surprisingly weak if force is applied. Depending on the direction of force application, the unbinding forces can more than triple. Our results suggest a model where multiple α-actinin/Z-repeat interactions cooperate to ensure long-term stable titin anchoring while allowing the individual components to exchange dynamically.

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.

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