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 (<0.3 nm) spatial resolution. Photo dam-age and sample heating; more complicated trapping configuration.

Two counterpropagating laser beams focused by two objectives enable a wide range of particles to be trapped with lower laser power than other traps. A movable micropipet is typically used to manipulate the opposite end of the sample to impose the stretching force.

Similar characteristics with single trap; wider range of particles can be trapped. Similar drawbacks as single trap; fixed optical trap position; complicated laser beam alignment.

By imposing a spatially encoded phase on the trapping laser beam, arbitrary light pat-terns can be formed in the specimen plane. This permits arrays of hundreds of optical traps that can rotate asymmetric particles and be moved or reconfigured under com-puter control.

Parallel processing of multiple beads; rotation of opti-cally trapped objects; arbitrary trap configurations. Photodamage and sample heating; difficult set-up and operations; slow reconfiguration (~10 Hz); decreased force and position resolution.

Cell sorting; mechanical manipulation of cell and lipid membranes.

Torque can be applied to a trapped birefrin-gent particle by rotating the polarization of the trapping laser. Birefringent particles that have been used to apply torque in the optical torque wrench include µm-scale fabri-cated quartz cylinders and crushed vaterite particles.

High precision control of torque and force; high spatial and temporal resolution. Photodamage and sample heating; difficult to maintain constant force; instrumen-tally challenging.

DNA topology and mechanical prop-erties of nucleosomes.

Two permanent magnets apply force and rotation on DNA attached to a superparamag-netic particle. The three-dimensional position of the bead is obtained by video tracking. The torque on the bead is sufficient in that it follows the rotation of the external magnets in a one-to-one correspondence.

Magnetic tweezers (MT); spatial resolution (1–5nm); temporal resolution (~100 Hz); force range (0.01–300 pN); passive force clamp. Lower resolution compared to OT and AFM; no 3D trapping; no torque measurement.

Manipulation of DNA topology. DNA topoisomerases and helicase assays.

Attaching a reporter “rotor” bead at a nick in the DNA allows rotation of the DNA to be directly observed. Force is applied on a superparamagnetic bead at the distal end of the DNA. Rotation and distortion of the DNA proximal to the rotor bead can be measured by the motion of the rotor bead. The torque on the DNA can be inferred from the rotation rate of the bead.

Spatial resolution (1–5 nm); temporal resolution (~300 Hz); force range (0.01–100 pN); torque sensitive; angular resolution (~10°). Lower resolution compared to OT and AFM; no 3D trapping.

Torque-dependent DNA structural transitions. Measurement of super-coiling activity of DNA gyrase.

Using a cylindrical magnet with a small mag-net attached on the side introduces a weak asymmetry in the circularly symmetric field along the axial direction that provides a small torque and permits precise torque measure-ments. Rotation of the bead is measured by tracking the position of a small nonmagnetic reporter bead attached to the magnetic bead.

Spatial resolution (1–10 nm), temporal resolution (0.1–0.01 s–1); force range (10–2–100 pN). Passive force clamp; torque sensitive; angular resolution (~0.1°). Low resolution compared to OT and AFM; no 3D trapping.

Torsional compliance of a RecA nucleoprotein filament.

Using a single magnet permits the applica-tion of force without torque. Torque can be applied by using a shape-asymmetric particle to break symmetry or by externally applying rotating magnetic field using electromagnets. Rotation of the bead is measured by tracking the position of a small nonmagnetic reporter bead attached to the magnetic bead.

Spatial resolution (1–10 nm), temporal resolution (10–1~0.01 s–1), stiffness (10–3~10–6 pN/nm); force range (0.01–100 pN); passive force clamp; torque sensitive. Low resolution compared to OT and AFM; no 3D trapping; no rotational control.

Measurement of rotation by RNA polymerase transcribing DNA. Mea-surement of rotation associated with polymerization of Rad51 on DNA.

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Vertical

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Single-beam gradient trap

Dual-beam (dumbbell) trap

Counter-propagating tweezers

Holographic OT (HOT)

Optical torque wrench

Dipole MT

Rotor bead MT

Magnetic torque tweezers

Single-pole MT (FOMT)

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