Temperature Effects on Optical Trapping Stability

This research was funded by the Ministerio de Ciencia e Innovacion de Espana (PID2019-106211RB-I00 and PID2019-105195RA-I00) and by Universidad Autonoma de Madrid and Comunidad Autonoma de Madrid (SI1/PJI/2019-00052). D.L. acknowledges a scholarship from the China Scholarship Council (No. 201808350097).In recent years, optically trapped luminescent particles have emerged as a reliable probe for contactless thermal sensing because of the dependence of their luminescence on environmental conditions. Although the temperature effect in the optical trapping stability has not always been the object of study, the optical trapping of micro/nanoparticles above room temperature is hindered by disturbances caused by temperature increments of even a few degrees in the Brownian motion that may lead to the release of the particle from the trap. In this report, we summarize recent experimental results on thermal sensing experiments in which micro/nanoparticles are used as probes with the aim of providing the contemporary state of the art about temperature effects in the stability of potential trapping processes.Spanish Government PID2019-106211RB-I00 PID2019-105195RA-I00Universidad Autonoma de Madrid and Comunidad Autonoma de Madrid SI1/PJI/2019-00052China Scholarship Council 20180835009

Probing DNA-Induced Colloidal Interactions and Dynamics with Scanning-Line Optical Tweezers

A promising route to forming novel nanoparticle-based materials is directed self-assembly, where the interactions among multiple species of suspended particles are intentionally designed to favor the self-assembly of a specific cluster arrangement or nanostructure. DNA provides a natural tool for directed particle assembly because DNA double helix formation is chemically specific — particles with short single-stranded DNA grafted on their surfaces will be bridged together only if those strands have complementary base sequences. Moreover, the temperature-dependent stability of such DNA bridges allows the resulting attraction to be modulated from negligibly weak to effectively irreversible over a convenient range of temperatures. Surprisingly, existing models for DNA-induced particle interactions are typically in error by more than an order of magnitude, which has hindered efforts to design complex temperature, sequence and time-dependent interactions needed for the most interesting applications. Here we report the first spatially resolved measurements of DNA-induced interactions between pairs of polystyrene microspheres at binding strengths comparable to those used in self-assembly experiments. The pair-interaction energies measured with our optical tweezers instrument can be modeled quantitatively with a conceptually straightforward and numerically tractable model, boding well for their application to direct self-assembly. In addition to understanding the equilibrium interactions between DNA-labeled particles, it is also important to consider the dynamics with which they bind to and unbind from one another. Here we demonstrate for the first time that carefully designed systems of DNA-functionalized particles exhibit effectively diffusion-limited binding, suggesting that these interactions are suitable to direct efficient self-assembly. We systematically explore the transition from diffusion-limited to reaction-limited binding by decreasing the DNA labeling density, and develop a simple dynamic model that is able to reproduce some of the anomalous kinetics observed in multivalent binding processes. Specifically, we find that when compounded, static disorder in the melting rate of single DNA duplexes gives rise to highly non-exponential lifetime distributions in multivalent binding. Together, our findings motivate a nanomaterial design approach where novel functional structures can be found computationally and then reliably realized in experiment

Nanoalignment by Critical Casimir Torques

The manipulation of microscopic objects requires precise and controllable forces and torques. Recent advances have led to the use of critical Casimir forces as a powerful tool, which can be finely tuned through the temperature of the environment and the chemical properties of the involved objects. For example, these forces have been used to self-organize ensembles of particles and to counteract stiction caused by Casimir-Liftshitz forces. However, until now, the potential of critical Casimir torques has been largely unexplored. Here, we demonstrate that critical Casimir torques can efficiently control the alignment of microscopic objects on nanopatterned substrates. We show experimentally and corroborate with theoretical calculations and Monte Carlo simulations that circular patterns on a substrate can stabilize the position and orientation of microscopic disks. By making the patterns elliptical, such microdisks can be subject to a torque which flips them upright while simultaneously allowing for more accurate control of the microdisk position. More complex patterns can selectively trap 2D-chiral particles and generate particle motion similar to non-equilibrium Brownian ratchets. These findings provide new opportunities for nanotechnological applications requiring precise positioning and orientation of microscopic objects

Methods of single-molecule energy landscape reconstruction with optical traps

Optical traps facilitate measurement of force and position as single molecules of DNA, RNA, or protein are unfolded and refolded. The effective energy landscape of a biomolecule can be reconstructed from the force and position data, providing insight into its structure and regulatory functions. We have developed new experimental and analytical methods to reconstruct energy landscapes by taking advantage of the harmonic constraint of an optical trap. We demonstrate the effectiveness of these methods using a model DNA hairpin and then apply these methods to study problems of practical biophysical interest. CCR5 mRNA has been demonstrated to stimulate -1 programmed ribosomal frameshifting and we measure its structural properties. We measure the binding energy of a GA/AG tandem mismatch, one of many mismatches with unusual properties. We use our single-molecule methods to reproduce bulk measurements of the nearest-neighbor DNA base-pair free energy parameters and we consider possible refinements to the model. We also study an alternative method of measuring energy landscapes, Dynamic Force Spectroscopy (DFS), and conduct experiments on DNA quadruplexes to demonstrate the effectiveness of DFS with optical traps. Finally, we develop theory to elucidate the role of noise in optical trap measurements of energy landscapes

Maximally Stiffening Composites Require Maximally Coupled Rather Than Maximally Entangled Polymer Species

Polymer composites are ideal candidates for next generation biomimetic soft materials because of their exquisite bottom-up designability. However, the richness of behaviours comes at a price: the need for precise and extensive characterisation of material properties over a highly-dimensional parameter space, as well as a quantitative understanding of the physical principles underlying desirable features. Here we couple large-scale Molecular Dynamics simulations with optical tweezers microrheology to characterise the viscoelastic response of DNA-actin composites. We discover that the previously observed non-monotonic stress-stiffening of these composites is robust, yet tunable, in a broad range of the parameter space that spans two orders of magnitude in DNA length. Importantly, we discover that the most pronounced stiffening is achieved when the species are maximally coupled, i.e. have similar number of entanglements, and not when the number of entanglements per DNA chain is largest. We further report novel dynamical oscillations of the microstructure of the composites, alternating between mixed and bundled phases, opening the door to future investigations. The generic nature of our system renders our results applicable to the behaviour of a broad class of polymer composites.Comment: Accepted in Soft Matte

Calibration of Tethered Particle Motion Experiments

The Tethered Particle Motion (TPM) method has been used to observe and characterize a variety of protein-DNA interactions including DNA loping and transcription. TPM experiments exploit the Brownian motion of a DNA-tethered bead to probe biologically relevant conformational changes of the tether. In these experiments, a change in the extent of the bead’s random motion is used as a reporter of the underlying macromolecular dynamics and is often deemed sufficient for TPM analysis. However, a complete understanding of how the motion depends on the physical properties of the tethered particle complex would permit more quantitative and accurate evaluation of TPM data. For instance, such understanding can help extract details about a looped complex geometry (or multiple coexisting geometries) from TPM data. To better characterize the measurement capabilities of TPM experiments involving DNA tethers, we have carried out a detailed calibration of TPM magnitude as a function of DNA length and particle size. We also explore how experimental parameters such as acquisition time and exposure time affect the apparent motion of the tethered particle. We vary the DNA length from 200 bp to 2.6 kbp and consider particle diameters of 200, 490 and 970 nm. We also present a systematic comparison between measured particle excursions and theoretical expectations, which helps clarify both the experiments and models of DNA conformation

Single-molecule methods for an improved understanding of biophysical interactions: from fundamental biology to applied nanotechnology

The advent of single-molecule methods has greatly extended the scale at which we are able to probe natural systems. The information that can be gained by studying biological systems on a single-molecule scale, in the absence of ensemble averaging, provides an unprecedented amount of detail about molecular interactions in real-time. Single-molecule biophysical techniques have provided unique insights into the nature of protein-DNA interactions, and have allowed for the development of novel platforms to study nano-bio interactions. In this thesis, we will describe two main sets of experiments to explore molecular interactions at the single-molecule scale. We will focus on the study of protein-DNA interactions and also the interactions between biological molecules and synthetic nanoparticles, using a variety of single-molecule techniques. Protein-DNA interactions are essential to cellular processes, many of which require proteins to recognize a specific DNA target-site. This search process is well-documented for monomeric proteins, but not as well understood for systems that require dimerization or oligomerization at the target site for activity. We present a single-molecule study of the target-search mechanism of Protelomerase TelK, a recombinase-like protein that is only active as a dimer. Interestingly, we observe that TelK undergoes 1D diffusion on non-target DNA as a monomer, as expected, but becomes immobile on DNA as a dimer or oligomer despite the absence of its target site. We further show that TelK condenses non-target DNA upon dimerization, forming a tightly bound nucleo-protein complex. Together with simulations of dimer-active protein search, our results suggest a search model whereby monomers diffuse along DNA, and subsequently dimerize to form an active complex on target DNA. These results show that target-finding occurs faster than nonspecific dimerization at biologically relevant protein concentrations. This model may provide insights into the search mechanisms of proteins that are active as multimeric complexes for a more accurate and comprehensive model for the target-search process by sequence specific proteins (SSPs). In addition to studying the target-search process of protelomerase TelK, we have also studied the molecular mechanism of TelK activity at the target site. We attempt to capture the dynamics responsible for DNA hairpin formation by TelK, and we discuss the unique features of TelK-DNA interactions that contribute to the complexity of this process. Nanomaterials have unique optical, chemical and mechanical properties that make them useful in biological applications, acting as drug and gene delivery agents, electrical and optical sensors, and cell-signaling components. Although many tools exist to characterize both biomolecules and nanomaterials, these methods are currently unable to give a detailed picture of biomolecular structure at the nano-bio interface. As a result, local electronic properties, bioavailability, toxicological effects, and basic molecular structure and conformation of biomolecules on nanoparticles remain unclear. Single-Walled Nanotubes (SWNTs) are allotropes of carbon with a cylindrical nanostructure. Though SWNTs tend to form insoluble aggregates, sonicating SWNTs with DNA forms a DNA-SWNT complex that is soluble in water. Single-stranded DNA (ssDNA) is believed to form a helical structure on the SWNT surface. This DNA-SWNT complex is not only soluble in water and does not appear to be toxic to mammalian cells, but it is also uptaken by mammalian cells via endocytosis. Therefore, there is significant interest in understanding the mechanism of SWNT encapsulation by ssDNA. However, current experimental tools have been unable to probe the structure of biomolecules on the surface of nanomaterials. Consequently, little is known about the mechanism by which ssDNA wraps SWNT, and how biomolecules interact with the resulting DNA-SWNT structure. In order to extend the range of biochemical interactions that can be detected on a SWNT surface, we have developed a variety of experimental platforms to study biological interactions on SWNT surfaces by extending several well-established single-molecule biophysics techniques to the study of nano-bio interactions. By applying single-molecule techniques to the study of the nano-bio interface, we uncover changes in the expected behavior of biomolecules. These effects include cooperative DNA hybridization, changes in the accessibility of DNA to nuclease proteins, and protein deactivation on a SWNT surface. We also uncover details of the mechanism by which ssDNA wraps SWNT to form a biologically-compatible nanoparticle-biomolecule conjugate

Modeling and Experimental Techniques to Demonstrate Nanomanipulation With Optical Tweezers

The development of truly three-dimensional nanodevices is currently impeded by the absence of effective prototyping tools at the nanoscale. Optical trapping is well established for flexible three-dimensional manipulation of components at the microscale. However, it has so far not been demonstrated to confine nanoparticles, for long enough time to be useful in nanoassembly applications. Therefore, as part of this work we demonstrate new techniques that successfully extend optical trapping to nanoscale manipulation. In order to extend optical trapping to the nanoscale, we must overcome certain challenges. For the same incident beam power, the optical binding forces acting on a nanoparticle within an optical trap are very weak, in comparison with forces acting on microscale particles. Consequently, due to Brownian motion, the nanoparticle often exits the trap in a very short period of time. We improve the performance of optical traps at the nanoscale by using closed-loop control. Furthermore, we show through laboratory experiments that we are able to localize nanoparticles to the trap using control systems, for sufficient time to be useful in nanoassembly applications, conditions under which a static trap set to the same power as the controller is unable to confine a same-sized particle. Before controlled optical trapping can be demonstrated in the laboratory, key tools must first be developed. We implement Langevin dynamics simulations to model the interaction of nanoparticles with an optical trap. Physically accurate simulations provide a robust platform to test new methods to characterize and improve the performance of optical tweezers at the nanoscale, but depend on accurate trapping force models. Therefore, we have also developed two new laboratory-based force measurement techniques that overcome the drawbacks of conventional force measurements, which do not accurately account for the weak interaction of nanoparticles in an optical trap. Finally, we use numerical simulations to develop new control algorithms that demonstrate significantly enhanced trapping of nanoparticles and implement these techniques in the laboratory. The algorithms and characterization tools developed as part of this work will allow the development of optical trapping instruments that can confine nanoparticles for longer periods of time than is currently possible, for a given beam power. Furthermore, the low average power achieved by the controller makes this technique especially suitable to manipulate biological specimens, but is also generally beneficial to nanoscale prototyping applications. Therefore, capabilities developed as part of this work, and the technology that results from it may enable the prototyping of three-dimensional nanodevices, critically required in many applications