Improving Signal/Noise Resolution in Single-Molecule Experiments Using Molecular Constructs with Short Handles

Forns, Nuria; Lorenzo, S. de; Manosas, Maria; Hayashi, Kentaro; Huguet, Josep Maria; Ritort, Fèlix

doi:10.1016/j.bpj.2011.01.071

Cited by 75 publications

(122 citation statements)

References 31 publications

(52 reference statements)

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“…Instead of moving the stage perpendicular to the beam direction, it is possible to maintain a simple geometry for all extensions by moving the stage along the direction of the laser beam, thus applying tension to a surface-tethered molecule in the axial direction. [39][40][41][42][43][44] Applying an axial force to surface tethered beads combines the high-yield of surface tethering, a relatively simple apparatus, and the ability to measure short tethers and to probe extensions down to zero, which allows for straightforward drift correction. Another important advantage of short tethers is that they yield finer spatial resolution.…”

Section: Introductionmentioning

confidence: 99%

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Practical axial optical trapping

Mack

Schlingman

Regan

et al. 2012

Review of Scientific Instruments

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We describe a new method for calibrating optical trapping measurements in which tension is applied in the direction of the laser beam to a molecule tethered between a surface and an optically trapped bead. Specifically, we present a generally-applicable procedure for converting from the measured scattering intensity and the measured stage displacement to applied tension and bead-coverslip separation, using measurements of the light intensity scattered from an untethered, trapped bead. Our calibration accounts for a number of effects, including aberrations and the interference of forwardreflected bead-scattered light with the trapping beam. To demonstrate the accuracy of our method, we show measurements of the DNA force-versus-extension relation using a range of laser intensities, and show that these measurements match the expected extensible wormlike-chain (WLC) behavior. Finally, we also demonstrate a force-clamp, in which the tension in a tether is held fixed while the extension varies as a result of molecular events.

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Section: Introductionmentioning

confidence: 99%

“…Another important advantage of short tethers is that they yield finer spatial resolution. 43,44 In addition, with an axial geometry, it is relatively straightforward to implement a reliable feedback loop, which maintains the force at a fixed value, namely a force clamp.…”

Section: Introductionmentioning

confidence: 99%

Practical axial optical trapping

Mack

Schlingman

Regan

et al. 2012

Review of Scientific Instruments

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show abstract

“…Kinetics does instead differ in the two setups: the apparent coexistence rate is more than two times higher for DT setup than it is for ST setup, in agreement with previous findings on the kinetic rates for tethers of different lengths and traps of different stiffness. 26,29 The consistency of the thermodynamic results shows that even when the beads are very close, forces are still correctly measured in the DT setup.…”

Section: A Hopping Experimentsmentioning

confidence: 56%

“…Although the elasticity of dsDNA is well established for kilobase long tethers, several recent experiments 23,24,26 and atomistic simulations 30 argue that DNA could be much more flexible at shorter length scales. In Sec.…”

Section: B Elastic Fluctuationsmentioning

confidence: 99%

Counter-propagating dual-trap optical tweezers based on linear momentum conservation

Ribezzi-Crivellari

Huguet

Ritort

2013

Review of Scientific Instruments

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We present a dual-trap optical tweezers setup which directly measures forces using linear momentum conservation. The setup uses a counter-propagating geometry, which allows momentum measurement on each beam separately. The experimental advantages of this setup include low drift due to alloptical manipulation, and a robust calibration (independent of the features of the trapped object or buffer medium) due to the force measurement method. Although this design does not attain the highresolution of some co-propagating setups, we show that it can be used to perform different single molecule measurements: fluctuation-based molecular stiffness characterization at different forces and hopping experiments on molecular hairpins. Remarkably, in our setup it is possible to manipulate very short tethers (such as molecular hairpins with short handles) down to the limit where beads are almost in contact. The setup is used to illustrate a novel method for measuring the stiffness of optical traps and tethers on the basis of equilibrium force fluctuations, i.e., without the need of measuring the force vs molecular extension curve. This method is of general interest for dual trap optical tweezers setups and can be extended to setups which do not directly measure forces.

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“…The TS is the highest free energy sequence. This synthesis procedure is described in detail in [16].…”

Section: The Free Energy Landscapementioning

confidence: 99%

Bayesian Approach to the Determination of the Kinetic Parameters of DNA Hairpins Under Tension

Ribezzi-Crivellari¹,

Wagner²,

Ritort³

2021

JNMP

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Improving Signal/Noise Resolution in Single-Molecule Experiments Using Molecular Constructs with Short Handles

Cited by 75 publications

References 31 publications

Practical axial optical trapping

Practical axial optical trapping

Counter-propagating dual-trap optical tweezers based on linear momentum conservation

Bayesian Approach to the Determination of the Kinetic Parameters of DNA Hairpins Under Tension

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