High bandwidth optical force clamp for investigation of molecular motor motion

2013 ◽  
Vol 103 (15) ◽  
pp. 153703 ◽  
Author(s):  
Subhrajit Roychowdhury ◽  
Tanuj Aggarwal ◽  
Srinivasa Salapaka ◽  
Murti V. Salapaka
Keyword(s):  
Molecular Motor ◽  
Optical Force ◽  
High Bandwidth ◽  
Force Clamp

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 An Automated Two-Dimensional Optical Force Clamp for Single Molecule Studies

2002 ◽  
Vol 83 (1) ◽  
pp. 491-501 ◽  
Author(s):  
Matthew J. Lang ◽  
Charles L. Asbury ◽  
Joshua W. Shaevitz ◽  
Steven M. Block
Keyword(s):  
Single Molecule ◽  
Optical Force ◽  
Two Dimensional ◽  
Single Molecule Studies ◽  
Force Clamp

scholarly journals Probing the kinesin reaction cycle with a 2D optical force clamp

2003 ◽  
Vol 100 (5) ◽  
pp. 2351-2356 ◽  
Author(s):  
S. M. Block ◽  
C. L. Asbury ◽  
J. W. Shaevitz ◽  
M. J. Lang
Keyword(s):  
Optical Force ◽  
Reaction Cycle ◽  
Force Clamp

scholarly journals Passive All-Optical Force Clamp for High-Resolution Laser Trapping

2005 ◽  
Vol 95 (20) ◽  
Author(s):  
William J. Greenleaf ◽  
Michael T. Woodside ◽  
Elio A. Abbondanzieri ◽  
Steven M. Block
Keyword(s):  
High Resolution ◽  
Optical Force ◽  
Laser Trapping ◽  
All Optical ◽  
Force Clamp

Probing the Kinesin reaction cycle with a 2D optical force clamp

2003 ◽  
Author(s):  
C.L. Asbury ◽  
J.W. Shaevitz ◽  
M.J. Lang ◽  
S.M. Block
Keyword(s):  
Optical Force ◽  
Reaction Cycle ◽  
Force Clamp

scholarly journals Direct physical study of kinetochore–microtubule interactions by reconstitution and interrogation with an optical force clamp

Methods ◽  
2010 ◽  
Vol 51 (2) ◽  
pp. 242-250 ◽  
Author(s):  
Andrew D. Franck ◽  
Andrew F. Powers ◽  
Daniel R. Gestaut ◽  
Trisha N. Davis ◽  
Charles L. Asbury
Keyword(s):  
Optical Force ◽  
Physical Study ◽  
Force Clamp
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