Force Spectroscopy with Dual-Trap Optical Tweezers: Molecular Stiffness Measurements and Coupled Fluctuations Analysis

Dual-trap optical tweezers are often used in high-resolution measurements in single-molecule biophysics. Such measurements can be hindered by the presence of extraneous noise sources, the most prominent of which is the coupling of fluctuations along different spatial directions, which may affect any...

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Bibliographic Details
Published in:Biophysical journal 2012-11, Vol.103 (9), p.1919-1928
Main Authors: Ribezzi-Crivellari, M., Ritort, F.
Format: Article
Language:English
Subjects:
ADN
analysis of variance
Aparells i instruments científics
Biofísica
Biophysics
Calibration
Coupling (molecular)
Couplings
DNA
DNA - chemistry
Elasticitat
Elasticity
Espectroscòpia
Fluctuation
Measurement
Misalignment
Models, Theoretical
Molecules
Molècules
Optical Tweezers
Polymers
Polímers
Proteins and Nucleic Acids
Scientific apparatus & instruments
Scientific apparatus and instruments
spectroscopy
Spectrum analysis
Spectrum Analysis - instrumentation
Spectrum Analysis - methods
Stiffness
Tethers
Variance analysis
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Summary:Dual-trap optical tweezers are often used in high-resolution measurements in single-molecule biophysics. Such measurements can be hindered by the presence of extraneous noise sources, the most prominent of which is the coupling of fluctuations along different spatial directions, which may affect any optical tweezers setup. In this article, we analyze, both from the theoretical and the experimental points of view, the most common source for these couplings in dual-trap optical-tweezers setups: the misalignment of traps and tether. We give criteria to distinguish different kinds of misalignment, to estimate their quantitative relevance and to include them in the data analysis. The experimental data is obtained in a, to our knowledge, novel dual-trap optical-tweezers setup that directly measures forces. In the case in which misalignment is negligible, we provide a method to measure the stiffness of traps and tether based on variance analysis. This method can be seen as a calibration technique valid beyond the linear trap region. Our analysis is then employed to measure the persistence length of dsDNA tethers of three different lengths spanning two orders of magnitude. The effective persistence length of such tethers is shown to decrease with the contour length, in accordance with previous studies.
ISSN:0006-3495
1542-0086
DOI:10.1016/j.bpj.2012.09.022