DNA-tethered Beads Research Articles

Highly-Parallel Microfluidics-Based Force Spectroscopy on Single Cytoskeletal Motors.

Cytoskeletal motors transform chemical energy into mechanical work to drive essential cellular functions. Optical trapping experiments have provided crucial insights into the operation of these molecular machines under load. However, the throughput of such force spectroscopy experiments is typically limited to one measurement at a time. Here, a highly-parallel, microfluidics-based method that allows for rapid collection of force-dependent motility parameters of cytoskeletal motors with two orders of magnitude improvement in throughput compared to currently available methods is introduced. Tunable hydrodynamic forces to stepping kinesin-1 motors via DNA-tethered beads and utilize a large field of view to simultaneously track the velocities, run lengths, and interaction times of hundreds of individual kinesin-1 molecules under varying resisting and assisting loads are applied. Importantly, the 16 µm long DNA tethers between the motors and the beads significantly reduces the vertical component of the applied force pulling the motors away from the microtubule. The approach is readily applicable to other molecular systems and constitutes a new methodology for parallelized single-molecule force studies on cytoskeletal motors.

An optimized software framework for real-time, high-throughput tracking of spherical beads.

Numerous biophysical techniques such as magnetic tweezers, flow stretching assays, or tethered particle motion assays rely on the tracking of spherical beads to obtain quantitative information about the individual biomolecules to which these beads are bound. The determination of these beads' coordinates from video-based images typically forms an essential component of these techniques. Recent advances in camera technology permit the simultaneous imaging of many beads, greatly increasing the information that can be captured in a single experiment. However, computational aspects such as frame capture rates or tracking algorithms often limit the rapid determination of such beads' coordinates. Here, we present a scalable and open source software framework to accelerate bead localization calculations based on the CUDA parallel computing framework. Within this framework, we implement the Quadrant Interpolation algorithm in order to accurately and simultaneously track hundreds of beads in real time using consumer hardware. In doing so, we show that the scatter derived from the bead tracking algorithms remains close to the theoretical optimum defined by the Cramer-Rao Lower Bound. We also explore the trade-offs between processing speed, size of the region-of-interests utilized, and tracking bias, highlighting in passing a bias in tracking along the optical axis that has previously gone unreported. To demonstrate the practical application of this software, we demonstrate how its implementation on magnetic tweezers can accurately track (with ∼1 nm standard deviation) 228 DNA-tethered beads at 58 Hz. These advances will facilitate the development and use of high-throughput single-molecule approaches.

SmAP: Manipulating DNA by Ultrasound - Single-Molecules Go Acoustic

The ability to study individual biomolecules in vitro has greatly expanded our knowledge of biological systems. Existing single-molecule techniques, such as optical and magnetic tweezers or atomic force microscopy, allow manipulation of individual biomolecules like DNA. They suffer from either being technically challenging or they offer low experimental throughput. We invented a novel lab-on-a-chip method to exert controllable forces on multiple DNA molecules simultaneously using ultrasound: single molecule Acoustical Pushing (smAP).smAP consists of a resonator integrated into a micro-fabricated fluidic chip. An acoustical pressure gradient is created homogeneously throughout the sample enabling to exert forces on DNA-tethered beads. By changing the amplitude of the driving voltage the pressure gradient can be altered, allowing sensitive control of the force applied to the DNA molecules.This approach makes it possible to apply forces up to hundreds of picoNewtons homogeneously over an area of several millimeter squared, allowing multiplexing to an unprecedented level. We validate this novel single-molecule method by recording force-distance curves of DNA molecules, both double- and single-stranded, in the presence of DNA-binding proteins. The simplicity and low cost makes smAP a widely accessible tool for biophysicists.

Magnetization Properties of Superparamagnetic Beads

Magnetic beads are frequently used in molecular biology and biophysics. In particular, nanometer- to micrometer-sized magnetic beads are a key component in magnetic tweezers and enable the application of precisely calibrated forces and torques to individual molecules of interest. Interestingly, despite the extensive use of magnetic beads, the origin of some of their apparent magnetic properties remains controversial. While the forces exerted on the particles in magnetic tweezers are well understood and have been quantitatively modeled from first principles (Lipfert et al., Biophys J., 2009), the torques are less well understood. Application of torque in magnetic tweezers requires the magnetization of the beads to exhibit some amount of anisotropy, the exact mechanism, however, remains poorly understood. Two models have been put forward: one relies on a small ferromagnetic component in addition to a larger paramagnetic component, while the other model postulates an ‘easy’ magnetization axis. We have performed two separate experiments to address this issue. First, we used direct angle tracking (Lipfert et al., Rev. Sci. Instrum., 2011) of DNA-tethered beads held in conventional magnetic tweezers to determine the magnetic field dependence of the rotational trap stiffness. The results are well described by a model proposed by Pavone and coworkers (Normanno et al., PRA, 2004) and argue in favor of a weak axis model. Second, we probed beads tethered to rotating flagellar motors of fixed E. coli cells to determine the field-dependent angular speed. Again, the studies suggest that the beads possess an ‘easy’ magnetization axis. Further insight and understanding of the intrinsic magnetization properties is desirable to improve the sensitivity of single-molecule magnetic tweezers and will be important for a quantitative understanding and optimization of the torque exerted in magnetic tweezers.

Threading immobilized DNA molecules through a solid-state nanopore at >100 μs per base rate.

In pursuit of developing solid-state nanopore-based DNA sequencing technology, we have designed and constructed an apparatus that can place a DNA-tethered probe tip near a solid-state nanopore, control the DNA moving speed, and measure the ionic current change when a DNA molecule is captured and released from a nanopore. The probe tip's position is sensed and controlled by a tuning fork based feedback force sensor and a nanopositioning system. Using this newly constructed apparatus, a DNA strand moving rate of >100 μs/base or <1 nm/ms in silicon nitride nanopores has been accomplished. This rate is 10 times slower than by manipulating DNA-tethered beads using optical tweezers and 1000 times slower than free DNA translocation through solid-state nanopores reported previously, which provides enough temporal resolution to read each base on a tethered DNA molecule using available single-channel recording electronics on the market today. This apparatus can measure three signals simultaneously: ionic current through a nanopore, tip position, and tip vibrational amplitude during the process of a DNA molecule's capture and release by a nanopore. We show results of this apparatus for measuring λ DNA's capture and release distances and for current blockage signals of λ DNA molecules biotinylated with one end and with both ends tethered to a tip.

Highly Parallel Magnetic Tweezers for the Study of DNA-Protein Interactions

Single-molecule force-spectroscopy techniques, such as magnetic and optical tweezers, have become a powerful tool in the detailed investigation of the biophysical properties of DNA and its interaction with proteins. Typically, the truly single molecule nature of these experiments heavily limits data collection. However, the particle image based data analysis used in magnetic tweezers naturally lends itself to the simultaneous measurement of multiple, spatially separated, molecules.We will describe the technical challenges involved in performing these multiplexed magnetic tweezers measurements and present the solutions. These include, the importance of post-experiment rather than real-time tracking due to computation constraints, the rapid selection of singularly tethered beads, and limits on the density of DNA-tethered magnetic beads. As a solution to the latter we describe a novel method to create targeted, non-random immobilization of DNA-tethered magnetic beads in regular, high density, arrays through micro-contact printing of DNA end-binding labels. This technique can allow an order of magnitude increase in data throughput of magnetic tweezers experiments. We are able to demonstrate the use of these techniques to perform experiments on hundreds of potential DNA-tethered beads in parallel resulting in statistical data on the mechanical properties of the DNA.Having developed the technique we describe its application to investigating an essential process in the repair of DNA damage through homologous recombination. Specifically, we study the nucleation of RecA proteins onto DNA and the subsequent formation of nucleoprotein filaments. With nucleation occurring only once per molecule per experiment our multiplexed approach is ideally suited to provide insights into this process along with the potential to capture other statistically rare protein-DNA interactions.

New Tool for Single Molecule Manipulation: Optical Pushing

The ability to measure and manipulate single molecules has greatly advanced the field of biophysics. The existing techniques, such as Optical and Magnetic Tweezers, each have their advantages and limitations. We present a proof-of-concept for an Optical Pushing apparatus, in which we exert an optical force on DNA-tethered beads with a collimated laser beam. This approach makes use of the temporal versatility of the laser power that is directly related to the force exerted on the tethered beads. This allows for fast force ramps that can be used for dynamic force spectroscopy as well as measurements on multiple single molecules at the same time.

A method to track rotational motion for use in single-molecule biophysics

The double helical nature of DNA links many cellular processes such as DNA replication, transcription, and repair to rotational motion and the accumulation of torsional strain. Magnetic tweezers (MTs) are a single-molecule technique that enables the application of precisely calibrated stretching forces to nucleic acid tethers and to control their rotational motion. However, conventional magnetic tweezers do not directly monitor rotation or measure torque. Here, we describe a method to directly measure rotational motion of particles in MT. The method relies on attaching small, non-magnetic beads to the magnetic beads to act as fiducial markers for rotational tracking. CCD images of the beads are analyzed with a tracking algorithm specifically designed to minimize crosstalk between translational and rotational motion: first, the in-plane center position of the magnetic bead is determined with a kernel-based tracker, while subsequently the height and rotation angle of the bead are determined via correlation-based algorithms. Evaluation of the tracking algorithm using both simulated images and recorded images of surface-immobilized beads demonstrates a rotational resolution of 0.1°, while maintaining a translational resolution of 1-2 nm. Example traces of the rotational fluctuations exhibited by DNA-tethered beads confined in magnetic potentials of varying stiffness demonstrate the robustness of the method and the potential for simultaneous tracking of multiple beads. Our rotation tracking algorithm enables the extension of MTs to magnetic torque tweezers (MTT) to directly measure the torque in single molecules. In addition, we envision uses of the algorithm in a range of biophysical measurements, including further extensions of MT, tethered particle motion, and optical trapping measurements.

Quantitative Modeling and Optimization of Magnetic Tweezers

Magnetic tweezers are a powerful tool to manipulate single DNA or RNA molecules and to study nucleic acid-protein interactions in real time. Here, we have modeled the magnetic fields of permanent magnets in magnetic tweezers and computed the forces exerted on superparamagnetic beads from first principles. For simple, symmetric geometries the magnetic fields can be calculated semianalytically using the Biot-Savart law. For complicated geometries and in the presence of an iron yoke, we employ a finite-element three-dimensional PDE solver to numerically solve the magnetostatic problem. The theoretical predictions are in quantitative agreement with direct Hall-probe measurements of the magnetic field and with measurements of the force exerted on DNA-tethered beads. Using these predictive theories, we systematically explore the effects of magnet alignment, magnet spacing, magnet size, and of adding an iron yoke to the magnets on the forces that can be exerted on tethered particles. We find that the optimal configuration for maximal stretching forces is a vertically aligned pair of magnets, with a minimal gap between the magnets and minimal flow cell thickness. Following these principles, we present a configuration that allows one to apply ≥40 pN stretching forces on ≈1- μm tethered beads.

Multiplexed single-molecule measurements with magnetic tweezers

We present a method for performing multiple single-molecule manipulation experiments in parallel with magnetic tweezers. We use a microscope with a low magnification, and thus a wide field of view, to visualize multiple DNA-tethered paramagnetic beads and apply an optimized image analysis routine to track the three-dimensional position of each bead simultaneously in real time. Force is applied to each bead using an externally applied magnetic field. Since variations in the field parameters are negligible across the field of view, nearly identical manipulation of all visible beads is possible. However, we find that the error in the position measurement is inversely proportional to the microscope's magnification. To mitigate the increased error caused by demagnification, we have developed a strategy based on tracking multiple fixed beads. Our system is capable of simultaneously manipulating and tracking up to 34 DNA-tethered beads at 60 Hz with approximately 1.5 nm resolution and with approximately 10% variation in applied force.