Allard J. Katan

Highly Parallel Magnetic Tweezers by Targeted DNA Tethering

Single-molecule force-spectroscopy methods such as magnetic and optical tweezers have emerged as powerful tools for the detailed study of biomechanical aspects of DNA-enzyme interactions. As typically only a single molecule of DNA is addressed in an individual experiment, these methods suffer from a low data throughput. Here, we report a novel method for targeted, nonrandom immobilization of DNA-tethered magnetic beads in regular arrays through microcontact printing of DNA end-binding labels. We show that the increase in density due to the arrangement of DNA-bead tethers in regular arrays can give rise to a one-order-of-magnitude improvement in data-throughput in magnetic tweezers experiments. We demonstrate the applicability of this technique in tweezers experiments where up to 450 beads are simultaneously tracked in parallel, yielding statistical data on the mechanics of DNA for 357 molecules from a single experimental run. Our technique paves the way for kilo-molecule force spectroscopy experiments, enabling the study of rare events in DNA-protein interactions and the acquisition of large statistical data sets from individual experimental runs.

High-Speed AFM Reveals the Dynamics of Single Biomolecules at the Nanometer Scale

Atomic force microscopy allows visualization of biomolecules with nanometer resolution under physiological conditions. Recent advances have improved the time resolution of the technique from minutes to tens of milliseconds, meaning that it is now possible to watch single biomolecules in action in real time. Here, we review this development.

Ionic Permeability and Mechanical Properties of DNA Origami Nanoplates on Solid-State Nanopores

While DNA origami is a popular and versatile platform, its structural properties are still poorly understood. In this study we use solid-state nanopores to investigate the ionic permeability and mechanical properties of DNA origami nanoplates. DNA origami nanoplates of various designs are docked onto solid-state nanopores where we subsequently measure their ionic conductance. The ionic permeability is found to be high for all origami nanoplates. We observe the conductance of docked nanoplates, relative to the bare nanopore conductance, to increase as a function of pore diameter, as well as to increase upon lowering the ionic strength. The honeycomb lattice nanoplate is found to have slightly better overall performance over other plate designs. After docking, we often observe spontaneous discrete jumps in the current, a process which can be attributed to mechanical buckling. All nanoplates show a nonlinear current-voltage dependence with a lower conductance at higher applied voltages, which we attribute to a physical bending deformation of the nanoplates under the applied force. At sufficiently high voltage (force), the nanoplates are strongly deformed and can be pulled through the nanopore. These data show that DNA origami nanoplates are typically very permeable to ions and exhibit a number of unexpected mechanical properties, which are interesting in their own right, but also need to be considered in the future design of DNA origami nanostructures.

Condensin Smc2-Smc4 Dimers Are Flexible and Dynamic

Summary Structural maintenance of chromosomes (SMC) protein complexes, including cohesin and condensin, play key roles in the regulation of higher-order chromosome organization. Even though SMC proteins are thought to mechanistically determine the function of the complexes, their native conformations and dynamics have remained unclear. Here, we probe the topology of Smc2-Smc4 dimers of the S. cerevisiae condensin complex with high-speed atomic force microscopy (AFM) in liquid. We show that the Smc2-Smc4 coiled coils are highly flexible polymers with a persistence length of only ∼4 nm. Moreover, we demonstrate that the SMC dimers can adopt various architectures that interconvert dynamically over time, and we find that the SMC head domains engage not only with each other, but also with the hinge domain situated at the other end of the ∼45-nm-long coiled coil. Our findings reveal structural properties that provide insights into the molecular mechanics of condensin complexes.

Dynamics of nucleosomal structures measured by high-speed Atomic Force Microscopy

The accessibility of DNA is determined by the number, position, and stability of nucleosomes, complexes consisting of a core of 8 histone proteins with DNA wrapped around it. Since the structure and dynamics of nucleosomes affects essential cellular processes, they are the subject of many current studies. Here, high-speed atomic force microscopy is used to visualize dynamic processes in nucleosomes and tetrasomes (subnucleosomal structures that contain 4 rather than 8 histones in the protein core). Nucleosomes can spontaneously disassemble in a process (at a 1 second timescale). For tetrasomes, multiple dynamic phenomena are observed. For example, during disassembly the formation of a DNA loop (∼25 nm in length) is seen, which remains stable for several minutes. For intact tetrasomes, dynamics in the form of sliding and reversible hopping between stable positions along the DNA are observed. The data emphasize that tetrasomes are not merely static objects but highly dynamic. Since tetrasomes (in contrast to nucleosomes) can stay on the DNA during transcription, the observed tetrasome dynamics is relevant for an understanding of the nucleosomal dynamics during transcription. These results illustrate the diversity of nucleosome dynamics and demonstrate the ability of high speed AFM to characterize protein-DNA interactions.

Spatial conductivity mapping of unprotected and capped black phosphorus using microwave microscopy

Thin layers of black phosphorus present an ideal combination of a 2D material with a tunable direct bandgap and high carrier mobility. However the material suffers from degradation in ambient conditions due to an oxidation reaction which involves water, oxygen and light. We have measured the spatial profile of the conductivity on flakes of black phosphorus as a function of time using scanning microwave impedance microscopy. A microwave excitation (3 GHz) allows to image a conducting sample even when covered with a dielectric layer. We observe that on bare black phosphorus, the conductivity changes drastically over the whole surface within a day. We demonstrate that the degradation process is slowed down considerably by covering the material with a 10 nm layer of hafnium oxide. It is stable for more than a week, opening up a route towards stable black phosphorus devices in which the high dielectric constant of hafnium oxide can be exploited. Covering black phosphorus with a 15 nm boron nitride flake changes the degradation process qualitatively, it is dominated by the edges of the flake indicating a diffusive process and happens on the scale of days.

Distortion of DNA Origami on Graphene Imaged with Advanced TEM Techniques

While graphene may appear to be the ultimate support membrane for transmission electron microscopy (TEM) imaging of DNA nanostructures, very little is known if it poses an advantage over conventional carbon supports in terms of resolution and contrast. Microscopic investigations are carried out on DNA origami nanoplates that are supported onto freestanding graphene, using advanced TEM techniques, including a new dark-field technique that is recently developed in our lab. TEM images of stained and unstained DNA origami are presented with high contrast on both graphene and amorphous carbon membranes. On graphene, the images of the origami plates show severe unwanted distortions, where the rectangular shape of the nanoplates is significantly distorted. From a number of comparative control experiments, it is demonstrated that neither staining agents, nor screening ions, nor the level of electron-beam irradiation cause this distortion. Instead, it is suggested that origami nanoplates are distorted due to hydrophobic interaction of the DNA bases with graphene upon adsorption of the DNA origami nanoplates.

Performance of THz Components Based on Microstrip PECVD SiNx Technology

We present a performance analysis of passive THz components based on Microstrip transmission lines with a 2-μm-thin plasma-enhanced chemical vapor deposition grown silicon nitride (PECVD SiNx) dielectric layer. A set of thru-reflect-line calibration structures is used for basic transmission line characterizations. We obtain losses of 9 dB/mm at 300 GHz. Branchline hybrid couplers are realized that exhibit 2.5-dB insertion loss, 1-dB amplitude imbalance, and −26-dB isolation, in agreement with simulations. We use the measured center frequency to determine the dielectric constant of the PECVD SiNx, which yields 5.9. We estimate the wafer–to-wafer variations to be of the order of 1%. Directional couplers are presented which exhibit −12-dB transmission to the coupled port and −26 dB to the isolated port. For transmission lines with 5-μm-thin silicon nitride (SiNx), we observe losses below 4 dB/mm. The thin SiNx dielectric membrane makes the THz components compatible with scanning probe microscopy cantilevers allowing the application of this technology in on-chip circuits of a THz near-field microscope.