Direct observation of the formation of a CRISPR–Cas12a R-loop complex at the single-molecule level

2020 ◽  
Vol 56 (14) ◽  
pp. 2123-2126 ◽  
Author(s):  
Yang Cui ◽  
Yangchao Tang ◽  
Meng Liang ◽  
Qinghua Ji ◽  
Yan Zeng ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Direct Observation ◽  
Single Molecule ◽  
Single Molecule Level ◽  
System P ◽  
Single Molecule Manipulation

An optical tweezers-based single-molecule manipulation assay was developed to detect the formation of an R-loop complex in the CRISPR–Cas12a system.

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.

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 Direct Observation of MUTYH and the Cancer-Associated Y150C Variant Binding to OG: A Mismatches at the Single Molecule Level

2018 ◽  
Vol 114 (3) ◽  
pp. 82a-83a
Author(s):  
Shane R. Nelson ◽  
Andrea J. Lee ◽  
Scott D. Kathe ◽  
Thomas S. Hilzinger ◽  
April M. Averill ◽  
...  
Keyword(s):  
Direct Observation ◽  
Single Molecule ◽  
Single Molecule Level

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.

Direct observation of the wrapping/unwrapping of ssDNA around/from a SWCNT at the single-molecule level: towards tuning the binding mode and strength

Nanoscale ◽  
2018 ◽  
Vol 10 (39) ◽  
pp. 18586-18596 ◽  
Author(s):  
Zhandong Li ◽  
Yu Song ◽  
Aisen Li ◽  
Weiqing Xu ◽  
Wenke Zhang
Keyword(s):  
Direct Observation ◽  
Single Molecule ◽  
Force Spectroscopy ◽  
Binding Mode ◽  
Single Molecule Force Spectroscopy ◽  
Single Molecule Level ◽  
Long Chain

AFM-based single-molecule force spectroscopy is employed to quantify the interactions between long-chain ssDNA and SWCNT and monitor the dynamic wrapping/unwrapping processes of ssDNA around/from the CNT.

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 Single-molecule manipulation of double-stranded DNA using optical tweezers: Interaction studies of DNA with RecA and YOYO-1

Cytometry ◽  
1999 ◽  
Vol 36 (3) ◽  
pp. 200-208 ◽  
Author(s):  
Martin L. Bennink ◽  
Orlando D. Sch�rer ◽  
Roland Kanaar ◽  
Kumiko Sakata-Sogawa ◽  
Juleon M. Schins ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Double Stranded Dna ◽  
Interaction Studies ◽  
Single Molecule Manipulation

scholarly journals Real-Time RecA Filament Disassembly in the Presence of RecX Monitored using Single-Molecule Manipulation by Optical Tweezers

2015 ◽  
Vol 108 (2) ◽  
pp. 69a
Author(s):  
Georgii Pobegalov ◽  
Alexandr Alekseev ◽  
Anton Sabantsev ◽  
Alexey Melnikov ◽  
Mikhail Khodorkovskiy ◽  
...  
Keyword(s):  
Real Time ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Single Molecule Manipulation
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