Dual-trap Optical Tweezers Research Articles

Following Translation and Folding of Individual Proteins by Single Ribosomes in Real-Time using Optical Tweezers

Protein synthesis is directly linked to protein folding. Translation kinetics plays a crucial role in forming secondary structural elements that lead to tertiary structure formation. However, how these two processes are coordinated is still not fully understood.Single-molecule techniques are essential for the study of protein synthesis and subsequent folding and were used in the past (1), since these are asynchronous processes that are extremely difficult to be observed using ensemble methods.Here we show for the first time to our knowledge full synthesis of three distinct proteins (hTau40, DHFR and GFPem) studied with dual-trap optical tweezers at the single-molecule level, continuing our previously published work (2). Our study explicates the intricate relationship between a protein's amino acid sequence, its co-translational elongation rate and folding.We show how hydrophobic collapse, which is the first step in protein folding, correlates with the number of hydrophobic amino acids that emerge from the ribosomal tunnel during synthesis. We also find that translational pauses are caused mainly by consecutive proline residues and positively charged amino acids. Finally, we propose a model that predicts the translation rate at a certain applied force and estimates hydrophobic collapse nucleation sites (manuscript submitted).1.Katranidis, A., D. Atta, R. Schlesinger, K. H. Nierhaus, T. Choli-Papadopoulou, I. Gregor, M. Gerrits, G. Buldt, and J. Fitter. 2009. Angew. Chem. Intl. ed 48, 1758-17612.Katranidis, A., W. Grange, R. Schlesinger, T. Choli-Papadopoulou, D. Bruggemann, M. Hegner, and G. Buldt. 2011. FEBS Lett. 585, 1859-1863

Measuring the interaction between a pair of emulsion droplets using dual-trap optical tweezers

Force–separation curves measured from a singe pair of emulsion droplets as a function of salt concentration (fits are DLVO theory).

Fast and Accurate Photodiode-Based Detection of Multiple Trap Optical Tweezers with Crosstalk-Elimination

Multiple trap optical tweezers have proven instrumental for a variety of exciting biophysical discoveries, e.g., from shedding light on molecular motor mechanisms to revealing intricate details of DNA-protein interactions. This ‘second’ generation of optical tweezers allows for advanced trapping geometries beyond single traps, however, these increased manipulation capabilities typically come at the price of more challenging position and force detection. The accuracy of position and force measurements is often compromised by crosstalk between the detected signals, this crosstalk leading to systematic and significant errors on the measured forces and distances. We developed an easy-to-implement simple method that enables simultaneous three-dimensional tracking of several individual objects in dual trap optical tweezers [1] and multi-trap holographic optical tweezers [2,3]. The method is based on spatial filtering and is highly compatible with standard back-focal-plane photodiode-based detection offering unrivaled temporal and spatial resolution with minimal crosstalk. This fast and accurate photodiode-based detection offers distinct advantages over camera-based solutions and opens the possibility for a variety of new biophysical assays. The reported technique significantly improves the accuracy of force-distance measurements, e.g., of single molecules, and hence provides much more scientific value for the experimental efforts.Publications:[1] D. Ott, S.N.S. Reihani, & L.B. Oddershede, “Crosstalk elimination in the detection of dual-beam optical tweezers by spatial filtering,” Review of Scientific Instruments,(2014).[2] D. Ott, S.N.S. Reihani, & L.B. Oddershede, “Simultaneous three-dimensional tracking of individual signals from multi-trap optical tweezers using fast and accurate photodiode detection,” Optics Express (2014).Filed Patent:[3] L.B. Oddershede, D. Ott, S.N.S. Reihani, “A detection system for an optical manipulation system for manipulating micro-particles or nano-particles of a sample by means of at least two optical traps,” PA 2014 70097, Denmark.

DNA-Induced Viral Assembly Studied in Real-Time by Optical Tweezers, Acoustic Force Spectroscopy and Atomic Force Microscopy

The simian vacuolating virus (SV40) can infect both human and apes and is of interest as capsule for drug delivery since it can efficiently infect a wide range of human cells. The icosahedral virus capsid is assembled from VP1 pentamers, and this assembly is stimulated by the presence of dsDNA. However, the real-time assembly process of SV40 and viruses in general is poorly understood limiting among other things our ability to manipulate capsid re-assembly in vitro for therapeutic purposes.Here, we show the real-time assembly of capsid around dsDNA employing dual-trap optical tweezers (OT). Protein assembly around the DNA leads to a drastic change in the contour length, which is readily observed when dsDNA is incubated with VP1 pentamers, indicating both kinking and crosslinking. Additionally, a new technology, acoustic force spectroscopy (AFS) capable to apply very low constant forces, provides indications of substantial capsid-like-structure formation observable as real-time shortening of DNA in the presence of VP1 combined with major rupture events when stretching the DNA-protein structure afterwards. In order to obtain visual information about protein coverage and arrangement on DNA, we generated atomic force microscope (AFM) images. In these images we observe significant cluster formation on DNA providing a good indication that we observe partial assembly. These results reveal that we can indeed track the assembly of viral capsids and visualize intermediates. This in turn provides a window into the complex kinetics of viral capsid formation.

Free-energy inference from partial work measurements in small systems.

Fluctuation relations (FRs) are among the few existing general results in nonequilibrium systems. Their verification requires the measurement of the total work performed on a system. Nevertheless in many cases only a partial measurement of the work is possible. Here we consider FRs in dual-trap optical tweezers where two different forces (one per trap) are measured. With this setup we perform pulling experiments on single molecules by moving one trap relative to the other. We demonstrate that work should be measured using the force exerted by the trap that is moved. The force that is measured in the trap at rest fails to provide the full dissipation in the system, leading to a (incorrect) work definition that does not satisfy the FR. The implications to single-molecule experiments and free-energy measurements are discussed. In the case of symmetric setups a second work definition, based on differential force measurements, is introduced. This definition is best suited to measure free energies as it shows faster convergence of estimators. We discuss measurements using the (incorrect) work definition as an example of partial work measurement. We show how to infer the full work distribution from the partial one via the FR. The inference process does also yield quantitative information, e.g., the hydrodynamic drag on the dumbbell. Results are also obtained for asymmetric dual-trap setups. We suggest that this kind of inference could represent a previously unidentified and general application of FRs to extract information about irreversible processes in small systems.

Optical tweezers reveal force plateau and internal friction in PEG-induced DNA condensation

The simplified artificial environments in which highly complex biological systems are studied do not represent the crowded, dense, salty, and dynamic environment inside the living cell. Consequently, it is important to investigate the effect of crowding agents on DNA. We used a dual-trap optical tweezers instrument to perform force spectroscopy experiments at pull speeds ranging from 0.3 to 270 μm/s on single dsDNA molecules in the presence of poly(ethylene glycol) (PEG) and monovalent salt. PEG of sizes 1,500 and 4,000 Da condensed DNA, and force-extension data contained a force plateau at approximately 1 pN. The level of the force plateau increased with increasing pull speed. During slow pulling the dissipated work increased linearly with pull speed. The calculated friction coefficient did not depend on amount of DNA incorporated in the condensate, indicating internal friction is independent of the condensate size. PEG300 had no effect on the dsDNA force-extension curve. The force plateau implies that condensation induced by crowding agents resembles condensation induced by multivalent cations.

Single Molecule Force Measurements of DNA and RNA Hairpin Structures

DNA and RNA hairpins contribute to fundamental mechanisms of molecular biology, for instance to the regulation of transcription and translation. Moreover, the hairpin secondary structure is a model system for studying the dynamic assembly of nucleic acid structures. Using a high-precision dual-trap optical tweezers setup, we performed a quantitative investigation of the folding dynamics of hairpins under mechanical load.A hairpin structure is composed of a double-helical stem and a single-stranded loop. Two different hairpins were studied, each one as a DNA and as an RNA molecule. They have the same stem of 13 basepairs, but exhibit loops of 10 and 18 nucleotides respectively. Significant differences are observed, not only between the two hairpins but also between DNA and RNA. As illustrated in the figure, hysteresis between unfolding and refolding curves is much stronger for RNA than for DNA. For narrow-loop hairpins, flipping between folded and unfolded states is observed for DNA and RNA. The wide-loop hairpins, however, show flips only for DNA. Altogether, our results show that the RNA molecule is more easily driven out-of-equilibrium by an external perturbation than DNA.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Optical tweezers technique and its applications

Since their advent in the 1980s, optical tweezers have attracted more and more attention due to their unique non-contact and non-invasion characteristics and their wide applications in physics, biology, chemistry, medical science and nanoscience. In this paper, we introduce the basic principle, the history and typical applications of optical tweezers and review our recent experimental works on the development and application of optical tweezers technique. We will discuss in detail several technological issues, including high precision displacement and force measurement in single-trap and dual-trap optical tweezers, multi-trap optical tweezers with each trap independently and freely controlled by means of space light modulator, and incorporation of cylindrical vector optical beams to build diversified optical tweezers beyond the conventional Gaussian-beam optical tweezers. We will address the application of these optical tweezers techniques to study biophysical problems such as mechanical deformation of cell membrane and binding energy between plant microtubule and microtubule associated proteins. Finally we present application of the optical tweezers technique for trapping, transporting, and patterning of metallic nanoparticles, which can be harnessed to manipulate surface plasmon resonance properties of these nanoparticles.

Three-dimensional light-scattering and deformation of individual biconcave human blood cells in optical tweezers

For studying the elastic properties of a biconcave red blood cell using the dual-trap optical tweezers without attaching microbeads to the cell, we implemented a three-dimensional finite element simulation of the light scattering and cell's deformation using the coupled electromagnetic and continuum mechanics modules. We built the vector field of the trapping beams, the cell structure layout, the hyperelastic and viscoelastic cell materials, and we reinforced the constraints on the cell constant volume in the simulation. This computation model can be useful for studying the scattering and the other mechanical properties of the biological cells.

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

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 all-optical 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 high-resolution 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.

The dynamic pause-unpackaging state, an off-translocation recovery state of a DNA packaging motor from bacteriophage T4.

Tailed bacteriophages and herpes viruses use powerful ATP-driven molecular motors to translocate their viral genomes into a preformed capsid shell. The bacteriophage T4 motor, a pentamer of the large terminase protein (gp17) assembled at the portal vertex of the prohead, is the fastest and most powerful known, consistent with the need to package a ~170-kb viral genome in approximately 5 min. Although much is known about the mechanism of DNA translocation, very little is known about how ATP modulates motor-DNA interactions. Here, we report single-molecule measurements of the phage T4 gp17 motor by using dual-trap optical tweezers under different conditions of perturbation. Unexpectedly, the motor pauses randomly when ATP is limiting, for an average of 1 s, and then resumes translocation. During pausing, DNA is unpackaged, a phenomenon so far observed only in T4, where some of the packaged DNA is slowly released. We propose that the motor pauses whenever it encounters a subunit in the apo state with the DNA bound weakly and incorrectly. Pausing allows the subunit to capture ATP, whereas unpackaging allows scanning of DNA until a correct registry is established. Thus, the "pause-unpackaging" state is an off-translocation recovery state wherein the motor, sometimes by taking a few steps backward, can bypass the impediments encountered along the translocation path. These results lead to a four-state mechanochemical model that provides insights into the mechanisms of translocation of an intricately branched concatemeric viral genome.

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 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.

Studying Single Red Blood Cells Under a Tunable External Force by Combining Passive Microrheology with Raman Spectroscopy

The dynamic micromechanical and structural properties of single human red blood cells are studied using a combination of dual trap optical tweezers and confocal Raman spectroscopy. Such a combination permits us to show a direct relationship between the rheological properties and chemical structure conformation. The frequency dependence of the complex stiffness of the cells was measured using both one and two probe response functions under identical experimental conditions. Both the microrheology and Raman measurements were performed at different stretching forces applied to the cell. A detailed analysis of the auto- and cross-correlated probe motions allows exploring the local and overall viscoelastic properties of the cells over a controlled range of the deformations. The observed growth of the cell viscoelasticity with stretching was associated with structural changes in the cell membrane monitored via the Raman spectroscopy.

Combined versatile high-resolution optical tweezers and single-molecule fluorescence microscopy

Optical trapping and single-molecule fluorescence are two major single-molecule approaches. Their combination has begun to show greater capability to study more complex systems than either method alone, but met many fundamental and technical challenges. We built an instrument that combines base-pair resolution dual-trap optical tweezers with single-molecule fluorescence microscopy. The instrument has complementary design and functionalities compared with similar microscopes previously described. The optical tweezers can be operated in constant force mode for easy data interpretation or in variable force mode for maximum spatiotemporal resolution. The single-molecule fluorescence detection can be implemented in either wide-field or confocal imaging configuration. To demonstrate the capabilities of the new instrument, we imaged a single stretched λ DNA molecule and investigated the dynamics of a DNA hairpin molecule in the presence of fluorophore-labeled complementary oligonucleotide. We simultaneously observed changes in the fluorescence signal and pauses in fast extension hopping of the hairpin due to association and dissociation of individual oligonucleotides. The combined versatile microscopy allows for greater flexibility to study molecular machines or assemblies at a single-molecule level.

Force–Fluctuation Relation of a Single DNA Molecule

We observed transverse fluctuations of single DNA molecules by fluorescence microscopy. The end-to-end distance of DNA molecules was varied by using dual trap optical tweezers, and the force–fluctuation relation was experimentally obtained in wide ranges of the force regime. In strong force regime, the theory of a stretched worm-like chain with fixed both ends explains the experimental results. On the other hand, in the low force regime, the fluctuations approach the value for an ideal ring polymer. We introduce an interpolate formula for the force–fluctuation relation by considering strong and low force limits, which captures the overall trend of experimental results. The proposed force–fluctuation relation will be useful for quantitative discussions in various sectors of polymer physics and biological processes which involve the conformation change of DNA and other biopolymers, where the loading and the fluctuation are relevant factors.

Investigation of Molecular Level Stress-Strain Relationships in Systems of Entangled F-Actin by Combined Force-Measuring Optical Tweezers and Fluorescence Microscopy

Actin plays a major role in cell structure, cell motility, vesicle and organelle transport, and muscle contraction. Actin's ability to play these major roles is a direct consequence of the intricate relationship between stress and strain in a variety of filamentous actin (F-actin) networks. A thorough understanding of the unique stress-strain relationships in complex F-actin networks at the molecular-level is currently lacking despite the importance of such networks to fields such as biomimetic material engineering and cell biology. Here we develop novel single-molecule instrumentation that combines dual-trap force-measuring optical tweezers with fluorescence microscopy to enable simultaneous characterization of intermolecular forces and molecular dynamics within F-actin networks. This instrumentation is combined with a novel technique in which single F-actin “probes” are used to apply molecular-level strains and measure induced stress within entangled F-actin systems while the deformations and dynamics of surrounding fluorescent-labeled filaments are simultaneously imaged. Specifically, the dual-trap optical tweezers trap fluorescent-labeled, microsphere-conjugated probes and measure the force induced on the probe as it is moved through a network of selectively-labeled entangled F-actin by using a nanoprecison piezoelectric stage to move the sample chamber relative to the traps. Fluorescence microscopy is used simultaneously to image the dynamics of the moving probe as well as the surrounding labeled molecules subject to the applied strain. Specific bioconjugation of the fluorescent polystyrene microspheres to the ends of labeled F-actin is achieved by carbodiimide attachment of gelsolin to microspheres and combining gelsolin-coated microspheres with fluorescent phalloidin labeled actin. This powerful single-molecule technique allows simultaneous measurement of intermolecular forces and dynamics and deformations of single molecules, providing the much needed link between stress and strain at the molecular level in complex F-actin networks.

Dual-trap optical tweezers with real-time force clamp control

Single molecule force clamp experiments are widely used to investigate how enzymes, molecular motors, and other molecular mechanisms work. We developed a dual-trap optical tweezers instrument with real-time (200 kHz update rate) force clamp control that can exert 0-100 pN forces on trapped beads. A model for force clamp experiments in the dumbbell-geometry is presented. We observe good agreement between predicted and observed power spectra of bead position and force fluctuations. The model can be used to predict and optimize the dynamics of real-time force clamp optical tweezers instruments. The results from a proof-of-principle experiment in which lambda exonuclease converts a double-stranded DNA tether, held at constant tension, into its single-stranded form, show that the developed instrument is suitable for experiments in single molecule biology.

Perturbation between two traps in dual-trap optical tweezers

Dual-trap optical tweezers are widely used in biological sciences and many other areas. The two traps are generally considered to be independent of each other in most situations. But when they are close and even touch side by side, the influence may not be negligible. We calculate the optical force of dielectric spherical particles trapped in dual-trap optical tweezers by means of the discrete dipole approximation method and analyze the perturbation effect between the two traps. We find that the electric field does not change much when it is scattered by the trapped particles. As a result, the scattering light from one sphere has a little perturbation effect on the trapping force upon the other sphere. The perturbation mainly comes from the other trap and is thus affected by the distance between the two traps. In the experiment, the displacement of the sphere trapped in an immovable trap is observed when another trap is moving close. The experiment data of relative displacement match qualitatively with the calculation data.

The Force Production by the Depolymerization Activity of MCAK

During cell division the replicating chromosomes must be precisely arranged and separated polewards. Though many cellular processes involving motility require force generation by motor proteins, the chromosome movement is suggested to use the energy stored at plus ends of the microtubules to which kinetochores are attached. This energy is converted into the chromosome movement via passive couplers, whereas the role of the microtubule-based motors is supposed to be limited to the regulation of microtubule dynamics. The microtubule-depolymerizing kinesins, such as mitotic centromere-associated kinesin (MCAK), which is a founding member of kinesin-13 family, facilitate microtubule dynamics in a spindle and are required for the chromosome congression and segregation; however, the key question - whether the depolymerizing activity generates tension to pull the chromosomes - has remained unsolved. To probe the link between the generation of tension and the microtubule-disassembling activity of MCAK, we developed a single-molecule assay to examine the interaction between a single microtubule and multiple MCAK molecules under the fluorescence microscope equipped with the dual-trap optical tweezers. Here we show that the microtubule-disassembling activity of MCAK generates significant tension. The depolymerization force increases with the number of interacting MCAK molecules. These results provide a simple and attractive model for the generation of the driving force and the regulation of chromosome segregation in a spindle by the activity of MCAK.

Determining the binding mode and binding affinity constant of tyrosine kinase inhibitor PD153035 to DNA using optical tweezers

Accurately predicting binding affinity constant (KA) is highly required to determine the binding energetics of the driving forces in drug–DNA interactions. Recently, PD153035, brominated anilinoquinazoline, has been reported to be not only a highly selective inhibitor of epidermal growth factor receptor but also a DNA intercalator. Here, we use a dual-trap optical tweezers to determining KA for PD153035, where KA is determined from the changes in B-form contour length (L) of PD153035–DNA complex. Here, L is fitted using a modified wormlike chain model. We found that a noticeable increment in L in 1mM sodium cacodylate was exhibited. Furthermore, our results showed that KA=1.18(±0.09)×104 (1/M) at 23±0.5°C and the minimum distance between adjacent bound PD153035≈11bp. We anticipate that by using this approach we can determine the complete thermodynamic profiles due to the presence of DNA intercalators.