SnapShot: Force Spectroscopy and Single-Molecule Manipulation€¦ · Single-molecule manipulation...

See online version for legend and references.1168 Cell 153, May 23, 2013 ©2013 Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cell.2013.04.047

SnapShot: Force Spectroscopy and Single-Molecule ManipulationYeonee Seol and Keir C. NeumanLaboratory of Molecular Biophysics, NHLBI, National Institutes of Health, Bethesda, MD 20892, USA

TECHNIQUE GENERAL DESCRIPTION KEY CHARACTERISTICS APPLICATION EXAMPLES

The deflection of a cantilever is measured using a laser beam and position-sensing detector to obtain force and displacement for both imaging and force spectroscopy. In force spectroscopy, the cantilever is attached to one end of the protein or DNA sample while the other end is immobilized on the surface. The force and position of the tip are measured as the surface is moved away from the tip, providing the force-extension curve of the sample.

High spatial resolution (0.5–1 nm); temporal resolution (>1 KHz); high force range (10–104 pN); high loading rate (10–105 pN/nm). Low force resolution compared to OT; difficult to operate under constant force; specificity can be difficult to control.

High force and high loading rate pull-ing and interaction assays; protein unfolding dynamics; protein-ligand interactions; ligand-receptor interac-tions on live cells.

A high numerical aperture (NA) objective focuses a laser to a diffraction limited spot, creating an “optical spring.” Dielectric par-ticles (~µm sized beads, bacteria, organelles) experience a restoring force toward the focus. Polystyrene or silica beads used as “handles” attached to proteins or DNA permit precise measurements of force and displacement in addition to three-dimensional control over the position of the sample.

High spatial resolution (~1 nm) and temporal resolution (10–50 kHz) if combined with laser detection system; intermediate force range (0.1–100 pN). Photo damage and sample heating; difficult to maintain a constant force; sensitive to mechanical drift.

Load-dependent enzyme activity of motor proteins such as kinesins on microtubules, myosins on actin. DNA, RNA, and protein unfolding and mechanical properties; DNA and RNA polymerases; DNA and RNA hecliases on nucleic acids.

Two independent single-beam traps are formed with one objective, and each end of the sample is held in one trap in a “dumbbell” geometry. Force is applied by moving the position of one of the traps relative to the other. The traps can be moved in the speci-men plane by steering the trapping laser with movable mirrors, acoustic optic deflectors, or electro optic deflectors.

Similar characteristics as single trap with potentially higher spatial resolution due to insensitivity to surface drift; base-pair (

SnapShot: Force Spectroscopy and Single-Molecule ManipulationYeonee Seol and Keir C. NeumanLaboratory of Molecular Biophysics, NHLBI, National Institutes of Health, Bethesda, MD 20892, USA

Single-molecule manipulation and force spectroscopy have become indispensable biophysical tools, permitting manipulation and measurement of individual biomolecules at previously unimaginable levels of precision. In this SnapShot, we present three single-molecule force spectroscopy techniques widely employed in biological science: atomic force spectroscopy, optical trapping, and magnetic tweezers. Due to space limitations, we focused on these three techniques most commonly used for single-molecule manipu-lation. Further details can be found in recent reviews of individual techniques.

ACKNOwLEDGMENTS

This research was supported by the Intramural Research Programs of the National Heart, Lung, and Blood Institute, National Institutes of Health.

REFERENCES

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1168.e1 Cell 153, May 23, 2013 ©2013 Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cell.2013.04.047

http://dx.doi.org/10.1016/j.cell.2013.04.047

SnapShot: Force Spectroscopy andSingle-Molecule ManipulationAcknowledgmentsReferences