Optical tweezers in single-molecule experiments

Zaltron, Annamaria; Merano, Michele; Mistura, Giampaolo; Sada, Cinzia; Seno, Flavio

doi:10.1140/epjp/s13360-020-00907-6

Cited by 38 publications

(28 citation statements)

References 130 publications

(172 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The experiments are carried out using a miniaturized dual-beam setup [ 47 , 48 , 49 ]. Briefly, two tightly focused counter-propagating laser beams (P = 200 mW,

= 845 nm) create a single optical trap, modelled as an harmonic potential ( Figure 1 a).…”

Section: Methodsmentioning

confidence: 99%

Sugar-Pucker Force-Induced Transition in Single-Stranded DNA

Viader-Godoy

Manosas

Ritort

2021

IJMS

View full text Add to dashboard Cite

The accurate knowledge of the elastic properties of single-stranded DNA (ssDNA) is key to characterize the thermodynamics of molecular reactions that are studied by force spectroscopy methods where DNA is mechanically unfolded. Examples range from DNA hybridization, DNA ligand binding, DNA unwinding by helicases, etc. To date, ssDNA elasticity has been studied with different methods in molecules of varying sequence and contour length. A dispersion of results has been reported and the value of the persistence length has been found to be larger for shorter ssDNA molecules. We carried out pulling experiments with optical tweezers to characterize the elastic response of ssDNA over three orders of magnitude in length (60–14 k bases). By fitting the force-extension curves (FECs) to the Worm-Like Chain model we confirmed the above trend:the persistence length nearly doubles for the shortest molecule (60 b) with respect to the longest one (14 kb). We demonstrate that the observed trend is due to the different force regimes fitted for long and short molecules, which translates into two distinct elastic regimes at low and high forces. We interpret this behavior in terms of a force-induced sugar pucker conformational transition (C3’-endo to C2’-endo) upon pulling ssDNA.

show abstract

“…The experiments are carried out using a miniaturized dual-beam setup [ 47 , 48 , 49 ]. Briefly, two tightly focused counter-propagating laser beams (P = 200 mW,

= 845 nm) create a single optical trap, modelled as an harmonic potential ( Figure 1 a).…”

Section: Methodsmentioning

confidence: 99%

Sugar-Pucker Force-Induced Transition in Single-Stranded DNA

Viader-Godoy

Manosas

Ritort

2021

IJMS

View full text Add to dashboard Cite

show abstract

“…Single molecule force spectroscopy (SMFS) has proven to be a powerful tool to investigate the properties of individual proteins, including mechanical stability [ 1 ], ligand-binding [ 2 ] and protein folding [ 3 , 4 ]. The three most commonly used methods are optical tweezers [ 4 , 5 ] (OT), atomic force microscopy [ 6 , 7 ] (AFM), and magnetic tweezers [ 8 , 9 ] (MT), which are able to measure forces in the piconewton range. While each method has its own features and limitations [ 10 ], it is typically required that the molecule under study is attached to a probe, i.e., an optically trapped bead, a tip of the AFM cantilever or a magnetic bead ( Figure 1 ).…”

Section: Introductionmentioning

confidence: 99%

“…While each method has its own features and limitations [ 10 ], it is typically required that the molecule under study is attached to a probe, i.e., an optically trapped bead, a tip of the AFM cantilever or a magnetic bead ( Figure 1 ). Background signals can be reduced by excluding non-specific short-range interactions between a surface and the protein of interest (POI) using a flexible linker between the protein and the trapped probe [ 5 ]. For optical tweezers, the linker additionally prevents the POI from being located directly in the path of the laser beam, avoiding thermal denaturation and accelerated photo-damage by the laser.…”

Section: Introductionmentioning

confidence: 99%

Bioconjugation Strategies for Connecting Proteins to DNA-Linkers for Single-Molecule Force-Based Experiments

Sleen

Tych

2021

Nanomaterials

View full text Add to dashboard Cite

The mechanical properties of proteins can be studied with single molecule force spectroscopy (SMFS) using optical tweezers, atomic force microscopy and magnetic tweezers. It is common to utilize a flexible linker between the protein and trapped probe to exclude short-range interactions in SMFS experiments. One of the most prevalent linkers is DNA due to its well-defined properties, although attachment strategies between the DNA linker and protein or probe may vary. We will therefore provide a general overview of the currently existing non-covalent and covalent bioconjugation strategies to site-specifically conjugate DNA-linkers to the protein of interest. In the search for a standardized conjugation strategy, considerations include their mechanical properties in the context of SMFS, feasibility of site-directed labeling, labeling efficiency, and costs.

show abstract

“…Single-molecule techniques have emerged to investigate the thermodynamics of individual proteins with high temporal resolution [9,10]. Force spectroscopy techniques such as Atomic Force Spectroscopy, Magnetic and Optical Tweezers have significantly contributed in achieving new insights into this research area [11,12]. Specifically, these methods use force as a "denaturant agent" to mechanically break the bonds that stabilize the protein structure [13].…”

Section: Introductionmentioning

confidence: 99%

Force Dependence of Proteins’ Transition State Position and the Bell–Evans Model

Rico-Pasto

Zaltron²,

Ritort

2021

Nanomaterials

Self Cite

View full text Add to dashboard Cite

Single-molecule force spectroscopy has opened a new field of research in molecular biophysics and biochemistry. Pulling experiments on individual proteins permit us to monitor conformational transitions with high temporal resolution and measure their free energy landscape. The force–extension curves of single proteins often present large hysteresis, with unfolding forces that are higher than refolding ones. Therefore, the high energy of the transition state (TS) in these molecules precludes kinetic rates measurements in equilibrium hopping experiments. In irreversible pulling experiments, force-dependent kinetic rates measurements show a systematic discrepancy between the sum of the folding and unfolding TS distances derived by the kinetic Bell–Evans model and the full molecular extension predicted by elastic models. Here, we show that this discrepancy originates from the force-induced movement of TS. Specifically, we investigate the highly kinetically stable protein barnase, using pulling experiments and the Bell–Evans model to characterize the position of its kinetic barrier. Experimental results show that while the TS stays at a roughly constant distance relative to the native state, it shifts with force relative to the unfolded state. Interestingly, a conversion of the protein extension into amino acid units shows that the TS position follows the Leffler–Hammond postulate: the higher the force, the lower the number of unzipped amino acids relative to the native state. The results are compared with the quasi-reversible unfolding–folding of a short DNA hairpin.

show abstract

Optical tweezers in single-molecule experiments

Cited by 38 publications

References 130 publications

Sugar-Pucker Force-Induced Transition in Single-Stranded DNA

Sugar-Pucker Force-Induced Transition in Single-Stranded DNA

Bioconjugation Strategies for Connecting Proteins to DNA-Linkers for Single-Molecule Force-Based Experiments

Force Dependence of Proteins’ Transition State Position and the Bell–Evans Model

Contact Info

Product

Resources

About