Dual-trap Optical Tweezers Research Articles

Rotating manipulation of femtosecond optical tweezers based on optical wedge-lens group.

Currently, research on optical tweezers technology predominantly focuses on single-trap optical tweezers, which have a limited controllable range. Multi-trap optical tweezers effectively address these limitations. This paper proposes a method for developing a dual-trap optical tweezers system utilizing basic optical elements. Two optical traps are created by reflecting a laser beam off the front and rear surfaces of a beam splitter. The transition between single-trap and dual-trap configurations is facilitated by a lens group, which allows for the adjustment of the distance between the two traps. Furthermore, by incorporating a rotatable optical wedge into the optical path, the optical trap can be rotated along an annular orbit of any radius. This study includes simulations and analyses of the effects of lens spacing, refractive index, and tilt angle on the rotational range of optical traps. An optical trapping experimental system was constructed, and its feasibility was demonstrated using polystyrene particles as the target objects.

Cell protrusions and contractions generate long-range membrane tension propagation

Membrane tension is thought to be a long-range integrator of cell physiology. Membrane tension has been proposed to enable cell polarity during migration through front-back coordination and long-range protrusion competition. These roles necessitate effective tension transmission across the cell. However, conflicting observations have left the field divided as to whether cell membranes support or resist tension propagation. This discrepancy likely originates from the use of exogenous forces that may not accurately mimic endogenous forces. We overcome this complication by leveraging optogenetics to directly control localized actin-based protrusions or actomyosin contractions while simultaneously monitoring the propagation of membrane tension using dual-trap optical tweezers. Surprisingly, actin-driven protrusions and actomyosin contractions both elicit rapid global membrane tension propagation, whereas forces applied to cell membranes alone do not. We present a simple unifying mechanical model in which mechanical forces that engage the actin cortex drive rapid, robust membrane tension propagation through long-range membrane flows.

Complexin-1 regulated assembly of single neuronal SNARE complex revealed by single-molecule optical tweezers

The dynamic assembly of the Synaptic-soluble N-ethylmaleimide-sensitive factor Attachment REceptor (SNARE) complex is crucial to understand membrane fusion. Traditional ensemble study meets the challenge to dissect the dynamic assembly of the protein complex. Here, we apply minute force on a tethered protein complex through dual-trap optical tweezers and study the folding dynamics of SNARE complex under mechanical force regulated by complexin-1 (CpxI). We reconstruct the clamp and facilitate functions of CpxI in vitro and identify different interplay mechanism of CpxI fragment binding on the SNARE complex. Specially, while the N-terminal domain (NTD) plays a dominant role of the facilitate function, CTD is mainly related to clamping. And the mixture of 1-83aa and CTD of CpxI can efficiently reconstitute the inhibitory signal identical to that the full-length CpxI functions. Our observation identifies the important chaperone role of the CpxI molecule in the dynamic assembly of SNARE complex under mechanical tension, and elucidates the specific function of each fragment of CpxI molecules in the chaperone process.

Studying PICH-induced nucleosome remodeling using dual-trap optical tweezers.

In anaphase, any unresolved DNA entanglements between the segregating sister chromatids can give rise to chromatin bridges. To prevent genome instability, chromatin bridges must be resolved prior to cytokinesis. The SNF2 protein PICH has been proposed to play a direct role in this process through the remodeling of nucleosomes. However, direct evidence of nucleosome remodeling by PICH has remained elusive. Here, we present an in vitro single-molecule assay that mimicks chromatin under tension, as is found in anaphase chromatin bridges. Applying a combination of dual-trap optical tweezers and fluorescence imaging of PICH and histones bound to a nucleosome-array construct, we show that PICH is a tension- and ATP-dependent nucleosome remodeler that facilitates nucleosome unwrapping and then subsequently slides remaining histones along the DNA. This work elucidates the role of PICH in chromatin-bridge dissolution, and might provide molecular insights into the mechanisms of related SNF2 proteins.

PICH acts as a force-dependent nucleosome remodeler

In anaphase, any unresolved DNA entanglements between the segregating sister chromatids can give rise to chromatin bridges. To prevent genome instability, chromatin bridges must be resolved prior to cytokinesis. The SNF2 protein PICH has been proposed to play a direct role in this process through the remodeling of nucleosomes. However, direct evidence of nucleosome remodeling by PICH has remained elusive. Here, we present an in vitro single-molecule assay that mimics chromatin under tension, as is found in anaphase chromatin bridges. Applying a combination of dual-trap optical tweezers and fluorescence imaging of PICH and histones bound to a nucleosome-array construct, we show that PICH is a tension- and ATP-dependent nucleosome remodeler that facilitates nucleosome unwrapping and then subsequently slides remaining histones along the DNA. This work elucidates the role of PICH in chromatin-bridge dissolution, and might provide molecular insights into the mechanisms of related SNF2 proteins.

Mechanical response of polyprotein revealed by single-molecule optical tweezers

We researched the mechanical unfolding of protein domains in monomeric protein NuG2 and the tandem polyproteins (NuG2)8 and (NuG2)16 using a dual-trap optical tweezers system. By stretching NuG2 and its polyproteins, (NuG2)8 and (NuG2)16 at the constant pulling speed of 500 nm s−1, we achieved the mechanical unfolding force of each domain in these proteins. Besides, we calculated the energy dissipation of NuG2, (NuG2)8 and (NuG2)16 by measuring the area enclosed by stretching and relaxation traces. Our results represent a key step towards engineering artificial polyproteins with controllable mechanical force and energy dissipation properties for force-buffering and energy dissipator applications.

Pich-induced nucleosome remodeling studied by dual-trap optical tweezers

In anaphase, entangled DNA between segregating sister chromatids can give rise to DNA threads known as ultrafine anaphase bridges (UFBs). To prevent genome instabilities, UFBs are resolved by a complex protein pathway. It has been proposed that a key step in UFB resolution is the remodeling of nucleosomes by the SNF2 protein PICH. However, direct evidence of nucleosome remodeling by PICH remains elusive. Here, we present a single-molecule assay that recapitulates chromatin re-organization during UFB resolution.

Generating Negatively Supercoiled DNA Using Dual-Trap Optical Tweezers.

Many genomic processes lead to the formation of underwound (negatively supercoiled) or overwound (positively supercoiled) DNA. These DNA topological changes regulate the interactions of DNA-binding proteins, including transcription factors, architectural proteins and topoisomerases. In order to advance our understanding of the structure and interactions of supercoiled DNA, we recently developed a single-molecule approach called Optical DNA Supercoiling (ODS). This method enables rapid generation of negatively supercoiled DNA (with between <5% and 70% lower helical twist than nonsupercoiled DNA) using a standard dual-trap optical tweezers instrument. ODS is advantageous as it allows for combined force spectroscopy, fluorescence imaging, and spatial control of the supercoiled substrate, which is difficult to achieve with most other approaches. Here, we describe how to generate negatively supercoiled DNA using dual-trap optical tweezers. To this end, we provide detailed instructions on the design and preparation of suitable DNA substrates, as well as a step-by-step guide for how to control and calibrate the supercoiling density produced.

Single Molecule Shape Determination of Biomolecules using DNA Nanoswitch Calipers

Single-molecule mechanical measurements can provide an unprecedented view into the shape, stabilities, and dynamics of biomolecules and their interactions. However, current challenges in determining the spatial arrangement of multiple residues within each molecule with a reasonable resolution and accuracy, have restricted applications. By combining structural DNA nanotechnology with dual-trap optical tweezers, we recently developed a mechanically reconfigurable DNA Nanoswitch Caliper (DNC) capable of measuring multiple distances on single biomolecules with atomic resolution.

Determinants for Fusion Speed of Biomolecular Droplets.

Biomolecular droplets formed through phase separation have a tendency to fuse. The speed with which fusion occurs is a direct indicator of condensate liquidity, which is key to both cellular functions and diseases. Using a dual-trap optical tweezers setup, we found the fusion speeds of four types of droplets to differ by two orders of magnitude. The order of fusion speed correlates with the fluorescence of thioflavin T, which in turn reflects the macromolecular packing density inside droplets. Unstructured protein or polymer chains pack loosely and readily rearrange, leading to fast fusion. In contrast, structured protein domains pack more closely and have to break extensive contacts before rearrangement, corresponding to slower fusion. This molecular interpretation for disparate fusion speeds provides mechanistic insight into the assembly and aging of biomolecular droplets.

Axial displacement calibration and tracking of optically trapped beads

High-precision axial displacement tracking of trapped beads is an indispensable feature of optical tweezers in advanced single-molecule studies. Here, we demonstrate an alternative method that enables axial calibration and tracking to be carried out on the same sample to avoid unnecessary errors. This method works by applying a dynamic force balance on a bead trapped between a piezoelectrically driven glass slide and an optical trap; in this configuration, the bead can be stopped precisely in different positions and imaged by a camera. A simple gradient algorithm is used to process the images into calibration data. After optimization of the calibration method and samples, our method exhibited better than 5 nm experimental axial resolution, with a measurement range of +/-500 nm around the objective focus at video speed. Moreover, for the first time, the deviation of the focusing plane in dual-trap optical tweezers was measured. We confirmed the axial deviation between two optical traps in our setup to be ~10 nm, corresponding to a force spectroscopy gage error of ~1 pN. This approach offers a favorable solution for in-use setup updating, as it can be seamlessly integrated into any optical tweezers system without requiring new hardware updates.

Constructing arrays of nucleosome positioning sequences using Gibson Assembly for single-molecule studies

As the basic building blocks of chromatin, nucleosomes play a key role in dictating the accessibility of the eukaryotic genome. Consequently, nucleosomes are involved in essential genomic transactions such as DNA transcription, replication and repair. In order to unravel the mechanisms by which nucleosomes can influence, or be altered by, DNA-binding proteins, single-molecule techniques are increasingly employed. To this end, DNA molecules containing a defined series of nucleosome positioning sequences are often used to reconstitute arrays of nucleosomes in vitro. Here, we describe a novel method to prepare DNA molecules containing defined arrays of the ‘601’ nucleosome positioning sequence by exploiting Gibson Assembly cloning. The approaches presented here provide a more accessible and efficient means to generate arrays of nucleosome positioning motifs, and facilitate a high degree of control over the linker sequences between these motifs. Nucleosomes reconstituted on such arrays are ideal for interrogation with single-molecule techniques. To demonstrate this, we use dual-trap optical tweezers, in combination with fluorescence microscopy, to monitor nucleosome unwrapping and histone localisation as a function of tension. We reveal that, although nucleosomes unwrap at ~20 pN, histones (at least histone H3) remain bound to the DNA, even at tensions beyond 60 pN.

Trapping and Manipulation of Single Cells in Crowded Environments.

Optical tweezers provide a powerful tool to trap and manipulate living cells, which is expected to help people gain physiological insights at single-cell level. However, trapping and manipulating single cells under crowded environments, such as blood vessels and lymph nodes, is still a challenging task. To overcome this issue, an annular beam formed by the far-field Bessel beam is introduced to serve as an optical shield to isolate the target cells from being disturbed. With this scheme, we successfully trapped and manipulated single blood cells in a crowded environment. Furthermore, we demonstrated manipulation of two lymphocytes ejected from a lymph node independently with dual-trap optical tweezers, which paves the way for exploring cell interactions under living conditions. Such technique might be helpful in the study of how natural killer cells response to virus-infected cells or cancer cells.

Supercoiling DNA optically

Cellular DNA is regularly subject to torsional stress during genomic processes, such as transcription and replication, resulting in a range of supercoiled DNA structures. For this reason, methods to prepare and study supercoiled DNA at the single-molecule level are widely used, including magnetic, angular-optical, micropipette, and magneto-optical tweezers. However, it is currently challenging to combine DNA supercoiling control with spatial manipulation and fluorescence microscopy. This limits the ability to study complex and dynamic interactions of supercoiled DNA. Here we present a single-molecule assay that can rapidly and controllably generate negatively supercoiled DNA using a standard dual-trap optical tweezers instrument. This method, termed Optical DNA Supercoiling (ODS), uniquely combines the ability to study supercoiled DNA using force spectroscopy, fluorescence imaging of the whole DNA, and rapid buffer exchange. The technique can be used to generate a wide range of supercoiled states, with between <5 and 70% lower helical twist than nonsupercoiled DNA. Highlighting the versatility of ODS, we reveal previously unobserved effects of ionic strength and sequence on the structural state of underwound DNA. Next, we demonstrate that ODS can be used to directly visualize and quantify protein dynamics on supercoiled DNA. We show that the diffusion of the mitochondrial transcription factor TFAM can be significantly hindered by local regions of underwound DNA. This finding suggests a mechanism by which supercoiling could regulate mitochondrial transcription in vivo. Taken together, we propose that ODS represents a powerful method to study both the biophysical properties and biological interactions of negatively supercoiled DNA.

In Situ Measurement of Depletion Caused by SDBS Micelles on the Surface of Silica Particles Using Optical Tweezers

Dual-trap optical tweezers have been used to directly measure the interaction forces between two silica particles upon controlling the concentration of the ionic surfactant sodium dodecylbenzenesulfonate (SDBS). By capturing two silica particles in one spot optical trap and one linear optical trap and controlling the linear trap to bring one particle to approach another sufficiently closer, the interaction forces between these two particles can be measured as the separation distance changes. Results showed that with increasing concentrations of SDBS, the interaction force between the two silica particles emerges at closer surface distance between two silica particles. Only repulsive force exists between silica particles below the critical micelle concentration (cmc) of SDBS and it could be well-fitted using the classical Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. However, the depletion attraction force appears above the cmc of SDBS which is induced by the generation of SDBS micelles. By in situ measurement of the interaction force between two silica particles in the presence of different concentrations of SDBS, the depletion force can be quantitatively calculated.

The Removal of Histones from Ultrafine Anaphase Bridges Studied by Optical Tweezers

During mitosis, duplicated DNA has to be faithfully segregated into two daughter cells. In anaphase, regions of entangled double-stranded DNA between segregating sister chromatids can give rise to DNA threads known as ultrafine anaphase bridges (UFBs). Failure to resolve UFBs can result in genome instabilities. It has been proposed that a key step in UFB resolution is the removal of histones, and that this is facilitated by the SNF2-family protein PICH. The mechanism of nucleosome remodeling by PICH has, however, remained elusive. Here, we present a single-molecule assay that recapitulates chromatin re-organization during UFB resolution. In vitro chromatin constructs consisting of 4 to 12 high affinity histone binding sites (601 sequences) within an 8.5 kbp DNA molecule were prepared using a combination of restriction-enzyme based cloning and Gibson assembly. After reconstitution with human histones, these constructs were manipulated within a multi-channel microfluidic flow cell using dual-trap optical tweezers. This approach allows us to measure tension-induced nucleosome unwrapping in the presence or absence of PICH. Our results suggest that, under moderate tension (10 pN), PICH facilitates the unwrapping of nucleosomes. In further experiments, we aim to correlate nucleosome stability with the dynamics of single fluorescently-labelled PICH proteins on DNA. Taken together, our single-molecule approach provides new insights into the mechanisms by which PICH is involved in anaphase UFB resolution.

Direct Measurement of π Coupling at the Single-Molecule Level using a Carbon Nanotube Force Sensor.

We report a carbon nanotube (CNT) force sensor that combines a suspended CNT transistor with dual-trap optical tweezers to explore the interactions between two individual molecules in the near-equilibrium regime with sub-piconewton resolution. The directly measured equilibrium force (1.2 ± 0.5 pN) is likely related to the binding force between a CNT and a single DNA base, where two aromatic rings spontaneously attract to each other due to the noncovalent forces between them. On the basis of our force measurements, the binding free energy per base is calculated (∼0.34 eV), which is in good agreement with theoretical simulations. Moreover, three-dimensional scanning photocurrent microscopy enables us to simultaneously monitor the morphology changes of the CNT, leading to a comprehensive reconstruction of the CNT-DNA binding dynamics. These experimental results shed light on the fundamental understanding of the mechanical coupling between CNTs and DNA molecules and, more importantly, provide a new platform for direct observation of intermolecular interfaces at the single-molecule level.

Two-point active microrheology in a viscous medium exploiting a motional resonance excited in dual-trap optical tweezers.

Two-point microrheology measurements from widely separated colloidal particles approach the bulk viscosity of the host medium more reliably than corresponding single-point measurements. In addition, active microrheology offers the advantage of enhanced signal to noise over passive techniques. Recently, we reported the observation of a motional resonance induced in a probe particle in dual-trap optical tweezers when the control particle was driven externally [Paul et al., Phys. Rev. E 96, 050102(R) (2017)2470-004510.1103/PhysRevE.96.050102]. We now demonstrate that the amplitude and phase characteristics of the motional resonance can be used as a sensitive tool for active two-point microrheology to measure the viscosity of a viscous fluid. Thus, we measure the viscosity of viscous liquids from both the amplitude and phase response of the resonance, and demonstrate that the zero crossing of the phase response of the probe particle with respect to the external drive is superior compared to the amplitude response in measuring viscosity at large particle separations. We compare our viscosity measurements with those using a commercial rheometer and obtain an agreement ∼1%. The method can be extended to viscoelastic material where the frequency dependence of the resonance may provide further accuracy for active microrheological measurements.

Full molecular trajectories of RNA polymerase at single base-pair resolution

In recent years, highly stable optical tweezers systems have enabled the characterization of the dynamics of molecular motors at very high resolution. However, the motion of many motors with angstrom-scale dynamics cannot be consistently resolved due to poor signal-to-noise ratio. Using an acousto-optic deflector to generate a "time-shared" dual-optical trap, we decreased low-frequency noise by more than one order of magnitude compared with conventional dual-trap optical tweezers. Using this instrument, we implemented a protocol that synthesizes single base-pair trajectories, which are used to test a Large State Space Hidden Markov Model algorithm to recover their individual steps. We then used this algorithm on real transcription data obtained in the same instrument to fully uncover the molecular trajectories of Escherichia coli RNA polymerase. We applied this procedure to reveal the effect of pyrophosphate on the distribution of dwell times between consecutive polymerase steps.

Translation and folding of single proteins in real time

Protein biosynthesis is inherently coupled to cotranslational protein folding. Folding of the nascent chain already occurs during synthesis and is mediated by spatial constraints imposed by the ribosomal exit tunnel as well as self-interactions. The polypeptide's vectorial emergence from the ribosomal tunnel establishes the possible folding pathways leading to its native tertiary structure. How cotranslational protein folding and the rate of synthesis are linked to a protein's amino acid sequence is still not well defined. Here, we follow synthesis by individual ribosomes using dual-trap optical tweezers and observe simultaneous folding of the nascent polypeptide chain in real time. We show that observed stalling during translation correlates with slowed peptide bond formation at successive proline sequence positions and electrostatic interactions between positively charged amino acids and the ribosomal tunnel. We also determine possible cotranslational folding sites initiated by hydrophobic collapse for an unstructured and two globular proteins while directly measuring initial cotranslational folding forces. Our study elucidates the intricate relationship among a protein's amino acid sequence, its cotranslational nascent-chain elongation rate, and folding.