Optical Tweezers Studies Research Articles

Biotinylation of reducing and non-reducing termini to create plug-and-play polysaccharides

Single-molecule studies continue to grow in popularity. In cases where biopolymer samples of interest exhibit variations in fine-structure between individual chains such single-molecule studies uniquely offer the promise of revealing deep structure-function relationships. Polysaccharides are typically studied in bulk and, as such, their study could greatly benefit from the application of single-molecule techniques. However, while for example single-molecule optical tweezers (OT) studies have become commonplace for DNA, studies of polysaccharides have lagged behind somewhat, complicated by the difficulty of studying molecules that amongst other things have more complex end-group chemistry. Recently, divalent streptavidin linkers have been shown to be capable of concatenating two pieces of biotin-terminated DNA to produce robust composite strings that run intact through conventional gels, and can be used in single-molecule OT experiments (Mohandas, Kent, Raudsepp, Jameson, & Williams, 2022). By using two such streptavidin linkers, biotin-terminated polymers could be inserted between two sections of DNA in order to facilitate single-molecule experiments on biopolymers that are currently difficult to address by other means. Here, we describe a generic approach for placing the required biotin moieties at both ends of polysaccharide chains, producing plug-and-play polysaccharide inserts that can be incorporated into composite polymer strings using streptavidin linking hubs.

Synthesis of Magneto-Controllable Polymer Nanocarrier Based on Poly(N-isopropylacrylamide-co-acrylic Acid) for Doxorubicin Immobilization.

In this work, the preparation procedure and properties of anionic magnetic microgels loaded with antitumor drug doxorubicin are described. The functional microgels were produced via the in situ formation of iron nanoparticles in an aqueous dispersion of polymer microgels based on poly(N-isopropylacrylamide-co-acrylic acid) (PNIPAM-PAA). The composition and morphology of the resulting composite microgels were studied by means of X-ray diffraction, Mössbauer spectroscopy, IR spectroscopy, scanning electron microscopy, atomic-force microscopy, laser microelectrophoresis, and static and dynamic light scattering. The forming nanoparticles were found to be β-FeO(OH). In physiological pH and ionic strength, the obtained composite microgels were shown to possess high colloid stability. The average size of the composites was 200 nm, while the zeta-potential was -27.5 mV. An optical tweezers study has demonstrated the possibility of manipulation with microgel using external magnetic fields. Loading of the composite microgel with doxorubicin did not lead to any change in particle size and colloidal stability. Magnetic-driven interaction of the drug-loaded microgel with model cell membranes was demonstrated by fluorescence microscopy. The described magnetic microgels demonstrate the potential for the controlled delivery of biologically active substances.

Optical microlever assisted DNA stretching.

Optical microrobotics is an emerging field that has the potential to improve upon current optical tweezer studies through avenues such as limiting the exposure of biological molecules of interest to laser radiation and overcoming the current limitations of low forces and unwanted interactions between nearby optical traps. However, optical microrobotics has been historically limited to rigid, single-body end-effectors rather than even simple machines, limiting the tasks that can be performed. Additionally, while multi-body machines such as microlevers exist in the literature, they have not yet been successfully demonstrated as tools for biological studies, such as molecule stretching. In this work we have taken a step towards moving the field forward by developing two types of microlever, produced using two-photon absorption polymerisation, to perform the first lever-assisted stretches of double-stranded DNA. The aim of the work is to provide a proof of concept for using optical micromachines for single molecule studies. Both styles of microlevers were successfully used to stretch single duplexes of DNA, and the results were analysed with the worm-like chain model to show that they were in good agreement.

From folding to function: complex macromolecular reactions unraveled one-by-one with optical tweezers.

Single-molecule manipulation with optical tweezers has uncovered macromolecular behaviour hidden to other experimental techniques. Recent instrumental improvements have made it possible to expand the range of systems accessible to optical tweezers. Beyond focusing on the folding and structural changes of isolated single molecules, optical tweezers studies have evolved into unraveling the basic principles of complex molecular processes such as co-translational folding on the ribosome, kinase activation dynamics, ligand-receptor binding, chaperone-assisted protein folding, and even dynamics of intrinsically disordered proteins (IDPs). In this mini-review, we illustrate the methodological principles of optical tweezers before highlighting recent advances in studying complex protein conformational dynamics - from protein synthesis to physiological function - as well as emerging future issues that are beginning to be addressed with novel approaches.

Optical tweezers study of membrane fluidity in small cell lung cancer cells.

The fluidity of the cell membrane is closely related to cancer metastasis/invasion. To test the relationship of membrane fluidity and invasiveness, we first demonstrated that transfection of small RNA miR-92b-3p can significantly increase invasiveness of the small cell lung cancer cell line SHP77. Then optical tweezers were used to measure membrane fluidity. This study employed continuous and step-like stretching methods to examine fluidity changes in SHP77 cell membranes before and after miR-92b-3p transfection. A newly developed physical model was used to derive the effective viscosity and static tension of the cell membrane from relaxation curves obtained via step-like stretching. Experiments showed that invasiveness and fluidity increased significantly after miR-92b-3p transfection. This study paved the way toward a better understanding of cancer cell invasion and membrane mechanical characteristics.

The Regulatory Function of the AAA4 ATPase Domain of Cytoplasmic Dynein

Cytoplasmic dynein is the primary motor for microtubule minus-end-directed transport and is indispensable to eukaryotic cells. Although each motor domain of dynein contains three active AAA+ ATPases (AAA1, 3, and 4), only the functions of AAA1 and 3 are known. Here, we use single-molecule fluorescence and optical tweezers studies to elucidate the role of AAA4 in dynein's mechanochemical cycle. We demonstrate that AAA4 controls the priming stroke of the motion-generating linker, which connects the dimerizing tail of the motor to the AAA+ ring.

Exploring the effects of approach velocity on depletion force and coalescence in oil-in-water emulsions.

An emulsion is a thermodynamically unstable system consisting of at least two immiscible liquid phases, one of which is dispersed in the other in the form of droplets of varying size. Most studies on emulsions have focused on the behaviour of emulsion droplets with diameter from ∼50 μm and upwards. However, the properties of smaller droplets may be highly relevant in order to understand the behaviour of emulsions, including their performance in numerous applications within the fields of food, industry, and medical science. The relatively long life-time and small size of these droplets compared to other emulsion droplets, make them suited for optical trapping and micromanipulation technologies. Optical tweezers have previously shown potential in the study of stabilized emulsions. Here we employ optical tweezers to examine unstable oil-in-water emulsions to determine the effects of system parameters on depletion force and coalescence times.

Munc13 binds and recruits SNAP25 to chaperone SNARE complex assembly.

Synaptic vesicle fusion is mediated by SNARE proteins-VAMP2 on the vesicle and Syntaxin-1/SNAP25 on the presynaptic membrane. Chaperones Munc18-1 and Munc13-1 cooperatively catalyze SNARE assembly via an intermediate 'template' complex containing Syntaxin-1 and VAMP2. How SNAP25 enters this reaction remains a mystery. Here, we report that Munc13-1 recruits SNAP25 to initiate the ternary SNARE complex assembly by direct binding, as judged by bulk FRET spectroscopy and single-molecule optical tweezer studies. Detailed structure-function analyses show that the binding is mediated by the Munc13-1 MUN domain and is specific for the SNAP25 'linker' region that connects the two SNARE motifs. Consequently, freely diffusing SNAP25 molecules on phospholipid bilayers are concentrated and bound in~1:1 stoichiometry by the self-assembled Munc13-1 nanoclusters.

The regulatory function of the AAA4 ATPase domain of cytoplasmic dynein

Cytoplasmic dynein is the primary motor for microtubule minus-end-directed transport and is indispensable to eukaryotic cells. Although each motor domain of dynein contains three active AAA+ ATPases (AAA1, 3, and 4), only the functions of AAA1 and 3 are known. Here, we use single-molecule fluorescence and optical tweezers studies to elucidate the role of AAA4 in dynein’s mechanochemical cycle. We demonstrate that AAA4 controls the priming stroke of the motion-generating linker, which connects the dimerizing tail of the motor to the AAA+ ring. Before ATP binds to AAA4, dynein remains incapable of generating motion. However, when AAA4 is bound to ATP, the gating of AAA1 by AAA3 prevails and dynein motion can occur. Thus, AAA1, 3, and 4 work together to regulate dynein function. Our work elucidates an essential role for AAA4 in dynein’s stepping cycle and underscores the complexity and crosstalk among the motor’s multiple AAA+ domains.

Force interactions between Yersiniae lipopolysaccharides and monoclonal antibodies: An optical tweezers study

This article reports the force spectroscopy investigation of interactions between lipopolysaccharides (LPSs) of two species from Yersinia genus and complementary (or heterologous) monoclonal antibodies (mAbs). We have obtained the experimental data by optical trapping on the “sensitized polystyrene microsphere – sensitized glass substrate” model system at its approach – retraction in vertical plane. We detected non-specific interactions in low-amplitude areas on histograms mainly due to physicochemical properties of abiotic surface and specific interactions in complementary pairs “antigen – antibodies” in high-amplitude areas (100–120 pN) on histograms. The developed measurement procedure can be used for detection of rupture forces in other molecular pairs.

Optical tweezers studies of transcription by eukaryotic RNA polymerases.

Transcription is the first step in the expression of genetic information and it is carried out by large macromolecular enzymes called RNA polymerases. Transcription has been studied for many years and with a myriad of experimental techniques, ranging from bulk studies to high-resolution transcript sequencing. In this review, we emphasise the advantages of using single-molecule techniques, particularly optical tweezers, to study transcription dynamics. We give an overview of the latest results in the single-molecule transcription field, focusing on transcription by eukaryotic RNA polymerases. Finally, we evaluate recent quantitative models that describe the biophysics of RNA polymerase translocation and backtracking dynamics.

Beyond the Hookean Spring Model: Direct Measurement of Optical Forces Through Light Momentum Changes.

The ability to measure forces in the range of 0.1-100 pN is a key feature of optical tweezers used for biophysical and cell biological studies. Analysis of the interactions between biomolecules and the forces that biomolecular motors generate at the single-molecule level has provided valuable insights in the molecular mechanisms that govern key cellular functions such as gene expression and the long-distance transport of organelles. Methods for determining the minute forces that biomolecular motors generate exhibit notable constraints that limit their application for studies other than the well-controlled in vitro experiments (although recent advances have been made that permit more quantitative optical tweezers studies insight living cells). One constraint comes from the linear approximation of the distance vs. force relationship used to extract the force from the position of the bead in the trap. This commonly employed "indirect" approach, although usually sufficiently precise, restricts the use of optical tweezers to a limited range of displacements (typically up to ±150nm for small beads). Measurements based on the detection of the light-momentum changes, on the other hand, offer a "direct" and precise way to determine forces even when the generated displacements reach the escape point, thus covering the complete force range developed by the trap. In this chapter, we detail the requirements for the design of a force-sensor instrument based on light-momentum changes using a high-numerical-aperture objective lens and provide insights into its construction. We further discuss the calibration of the system and the main steps for its routine operation.

Optical tweezers study of red blood cell aggregation and disaggregation in plasma and protein solutions

Kinetics of optical tweezers (OT)-induced spontaneous aggregation and disaggregation of red blood cells (RBCs) were studied at the level of cell doublets to assess RBC interaction mechanics. Measurements were performed under in vitro conditions in plasma and fibrinogen and fibrinogen + albumin solutions. The RBC spontaneous aggregation kinetics was found to exhibit different behavior depending on the cell environment. In contrast, the RBC disaggregation kinetics was similar in all solutions qualitatively and quantitatively, demonstrating a significant contribution of the studied proteins to the process. The impact of the study on assessing RBC interaction mechanics and the protein contribution to the reversible RBC aggregation process is discussed.

Fluctuations of a membrane nanotube revealed by high-resolution force measurements.

Pulling membrane nanotubes from liposomes presents a powerful method to gain access to membrane mechanics. Here we extend classical optical tweezers studies to infer membrane nanotube dynamics with high spatial and temporal resolution. We first validate our force measurement setup by accurately measuring the bending modulus of EPC membrane in tube pulling experiments. Then we record the position signal of a trapped bead when it is connected, or not, to a tube. We derive the fluctuation spectrum of these signals and find that the presence of a membrane nanotube induces higher fluctuations, especially at low frequencies (10-1000 Hz). We analyse these spectra by taking into account the peristaltic modes of nanotube fluctuations. This analysis provides a new experimental framework for a quantitative study of the fluctuations of nanotubular membrane structures that are present in living cells, and now classically used for in vitro biomimetic approaches.

Cell-scaffold adhesion dynamics measured in first seconds predicts cell growth on days scale – optical tweezers study.

Understanding the cell-biomaterial interface from the very first contact is of crucial importance for their successful implementation and function in damaged tissues. However, the lack of bio- and mechano-analytical methods to investigate and probe the initial processes on the interface, especially in 3D, raises the need for applying new experimental techniques. In our study, optical tweezers combined with confocal fluorescence microscopy were optimized to investigate the initial cell-scaffold contact and to investigate its correlation with the material-dependent cell growth. By the optical tweezers-induced cell manipulation accompanied by force detection up to 100 pN and position detection by fluorescence microscopy, accurate adhesion dynamics and strength analysis was implemented, where several attachment sites were formed on the interface in the first few seconds. More importantly, we have shown that dynamics of cell adhesion on scaffold surfaces correlates with cell growth on the days scale, which indicates that the first seconds of the contact could markedly direct further cell response. Such a contact dynamics analysis on 3D scaffold surfaces, applied for the first time, can thus serve to predict scaffold biocompatibility.

Mechanisms of HCV NS3 helicase monitored by optical tweezers.

As one of the essential enzymes for viral genome replication, the hepatitis C virus NS3 helicase is one of the best characterized RNA helicases to date in understanding the mechanistic cycles in a helicase-catalyzed strand separation reaction. Recently, single-molecule studies on NS3, in particular the use of optical tweezers with sub-base pair spatial resolution, have allowed people to examine the potential elementary steps of NS3 in unwinding the double-stranded RNA fueled by ATP binding and hydrolysis. In this chapter, I detail the essential technical elements involved in conducting a high-resolution optical tweezers study of NS3 helicase, starting from the purification of the recombinant helicase protein from E. coli to setting up a high-resolution single-molecule experiment using optical tweezers.

093 Ultra rapid warming of cryo samples using an IR laser pulse

Our laboratory has been investigating the effect of warming rate on survival of mouse oocytes after very rapid cooling. Cooling and warming rates are affected by sample thermal mass, surface area, thermal impedance, and delta T. To maximize these rates we use 0.1 ul samples placed on Cryotops and plunged directly into LN2 for cooling and into room temperature buffer for warming. The maximum warming rate obtained is ∼ 100,000 C/min and it yields the highest survivals. However, extrapolation of the experimental data suggests faster warming would yield higher survivals. Here we report on the use of an IR laser pulse to achieve warming rates 3.5 to 100 times faster. Our objective was to choose a laser wavelength which would warm the cryo buffer while leaving the cells ‘untouched’, thus leading to indirect warming of the cells. Optical tweezer studies show that a wide variety of cell types can tolerate very high intensities of 1064 nm IR energy emitted by Nd:YAG lasers and so this was the type chosen. Calculations indicate the laser intensity for cryo melting to be significantly less than that used for optical tweezers. Most cryo buffers, including the EAFS we use are relatively transparent in the IR. The buffer IR absorption is easily increased by adding a little India ink. India ink consists primarily of carbon particles from 0.1 to 1 um in diameter, and at the concentration we use, 0.25% (V/V), is harmless to the oocytes. The concentration is carefully chosen to allow most of the IR energy to pass completely through the sample. This yields relatively even sample heating from front to back. We estimate ink-free samples absorb ∼ 9% of the incident energy, and the added ink absorbs ∼ 25% of the remaining energy, yielding a total absorption of ∼ 30%. We have developed a small cryo ‘jig’ which lifts the sample out of an LN2 bath, covers the bath, and fires a laser pulse, all in less than one sec. This assures that the sample is rapidly warmed by the laser pulse rather than by slower air warming. Thus, the samples are warmed from ∼ −180°C to ∼ 0°C by the laser pulse. We have computed the required laser energy for melting, but there are so many variables that we have found it preferable to empirically determine the required laser energy to just melt the samples for a given experiment. This is done by direct visual observation with a 15 x stereo microscope built into the laser system. We use a 40 joule welding laser (for long pulse durations!) with a beam diameter of 2 mm and a pulse length of 0.5 to 30 msec. This yields warming rates from 3.5 x 10 5 to 2 x 10 7 C/min. However, modeling indicates that rates higher than 1 x 10 7 C/min yield unacceptably large thermal gradients across the 70 um diameter oocytes and are not used. Preliminary experiments at a warming rate of 3.5 x 10 5 C/min yield survivals comparable to our traditional methods. Source of Funding: NIH Grant 8R01 OD 011201, Peter Mazur, PI. E. Paredes FPU Research Stays Fellowship from the Spanish Government, 1 November 2012-31 January 2013. Conflict of Interest: None declared. fkleinha@iupui.edu

High-Bandwidth Magnetic Tweezers for Applying Torsion to Single DNA Molecules

The mechanical and functional properties of DNA arise from its double helical structure. It is now widely accepted that the torsional properties of DNA and DNA supercoiling play an important role in the kinetics of many DNA-binding proteins, but the mechanism underlying this relationship remains unclear. To address this gap in our understanding, we need an instrument that can accurately measure and control torsional stress applied to DNA. We have developed a high-bandwidth electromagnetic trapping system that can generate a uniform magnetic field in the sample region and apply constant torque above 102 pN·nm on the samples under study. The octupole magnetic trap is integrated into a microscope-based particle tracking system and can rotate superparamagnetic particles with three degrees of rotational freedom. The large signal bandwidth of the current in the coils can reach above 3kHz at 800uH inductive load and the heat generated by the current is dissipated by an active PID-controlled cooling system to prevent heating biological samples. The magnetic trap is being designed to independently control force and torque, allowing us to confine superparamagnetic particles in a trap with low torsional stiffness that is suitable for torque application and measurement at biologically relevant scales. To directly measure the torsional strain in DNA, we are planning to use superparamagnetic beads coated with metal on one hemisphere. Our magnetic torque tweezers are intended to quantitatively measure the changes of torsional stress in DNA and overcome the complexity and heating problems shared by previous optical and magnetic tweezers studies.

A novel two-layer, coupled finite element approach for modeling the nonlinear elastic and viscoelastic behavior of human erythrocytes

A novel finite element approach is presented to simulate the mechanical behavior of human red blood cells (RBC, erythrocytes). As the RBC membrane comprises a phospholipid bilayer with an intervening protein network, we propose to model the membrane with two distinct layers. The fairly complex characteristics of the very thin lipid bilayer are represented by special incompressible solid shell elements and an anisotropic viscoelastic constitutive model. Properties of the protein network are modeled with an isotropic hyperelastic third-order material. The elastic behavior of the model is validated with existing optical tweezers studies with quasi-static deformations. Employing material parameters consistent with literature, simulation results are in excellent agreement with experimental data. Available models in literature neglect either the surface area conservation of the RBC membrane or realistic loading conditions of the optical tweezers experiments. The importance of these modeling assumptions, that are both included in this study, are discussed and their influence quantified. For the simulation of the dynamic motion of RBC, the model is extended to incorporate the cytoplasm. This is realized with a monolithic fully coupled fluid-structure interaction simulation, where the fluid is described by the incompressible Navier-Stokes equations in an arbitrary Lagrangian Eulerian framework. It is shown that both membrane viscosity and cytoplasm viscosity have significant influence on simulation results. Characteristic recovery times and energy dissipation for varying strain rates in dynamic laser trap experiments are calculated for the first time and are found to be comparable with experimental data.

RNA Reactions One Molecule at a Time

Much of the dynamics information is lost in bulk measurements because of the population averaging. Single-molecule methods measure one molecule at a time; they provide knowledge not obtainable by other means. In this article, we review the application of the two most widely used single-molecule methods--fluorescence resonance energy transfer (FRET) and force versus extension measurements--to several RNA reactions. First, we discuss folding/unfolding studies on a hairpin ribozyme that revealed multiple conformations of the RNA with distinct kinetics, and on a series of RNA pseudoknots, whose mechanical stabilities were found to show a strong correlation with their frameshifting efficiency during translation. We also discuss several RNA-related molecular motors. Single-molecule experiments revealed detailed mechanisms for the interaction of HIV reverse transcriptase and nucleic acid helicases (NS3 and RIG-1) with their substrates. Optical tweezers studies showed that translation of a single messenger RNA by a ribosome occurs by successive translocation-and-pause cycles. Single-molecule FRET experiments yielded important information on ribosome conformational changes and tRNA dynamics during translation. Overall, single-molecule experiments have been very valuable for understanding RNA reactions.