Ring-shaped structural maintenance of chromosomes (SMC) complexes like condensin and cohesin extrude loops of DNA. It remains, however, unclear how they can extrude DNA loops in chromatin that is bound with proteins. Here, we use in vitro single-molecule visualization to show that nucleosomes, RNA polymerase, and dCas9 pose virtually no barrier to loop extrusion by yeast condensin. We find that even DNA-bound nanoparticles as large as 200 nm, much bigger than the SMC ring size, also translocate into DNA loops during extrusion by condensin and cohesin. This even occurs for a single-chain version of cohesin in which the ring-forming subunits are covalently linked and cannot open to entrap DNA. The data show that SMC-driven loop extrusion has surprisingly little difficulty in accommodating large roadblocks into the loop. The findings also show that the extruded DNA does not pass through the SMC ring (pseudo)topologically, hence pointing to a nontopological mechanism for DNA loop extrusion.

Chromosome inheritance depends on centromeres, epigenetically specified regions of chromosomes. While conventional human centromeres are known to be built of long tandem DNA repeats, much of their architecture remains unknown. Using single-molecule techniques such as AFM, nanopores, and optical tweezers, we find that human centromeric DNA exhibits complex DNA folds such as local hairpins. Upon binding to a specific sequence within centromeric regions, the DNA-binding protein CENP-B compacts centromeres by forming pronounced DNA loops between the repeats, which favor inter-chromosomal centromere compaction and clustering. This DNA-loop-mediated organization of centromeric chromatin participates in maintaining centromere position and integrity upon microtubule pulling during mitosis. Our findings emphasize the importance of DNA topology in centromeric regulation and stability.

Nucleoid-associated proteins (NAPs) are a class of highly abundant DNA-binding proteins in bacteria and archaea. While both the composition and relative abundance of the NAPs change during the bacterial growth cycle, surprisingly little is known about their crosstalk in mutually binding and stabilizing higher-order nucleoprotein complexes in the bacterial chromosome. Here, we use atomic force microscopy and solid-state nanopores to investigate long-range nucleoprotein structures formed by the binding of two major NAPs, FIS and H-NS, to DNA molecules with distinct binding site arrangements. We find that spatial organization of the protein binding sites can govern the higher-order architecture of the nucleoprotein complexes. Based on sequence arrangement the complexes differed in their global shape and compaction as well as the extent of FIS and H-NS binding. Our observations highlight the important role the DNA sequence plays in driving structural differentiation within the bacterial chromosome.

2D nanoslit devices, where two crystals with atomically flat surfaces are separated by only a few nanometers, have attracted considerable attention because their tunable control over the confinement allows for the discovery of unusual transport behavior of gas, water, and ions. Here, the passage of double-stranded DNA molecules is studied through nanoslits fabricated from exfoliated 2D materials, such as graphene or hexagonal boron nitride, and the DNA polymer behavior is examined in this tight confinement. Two types of events are observed in the ionic current: long current blockades that signal DNA translocation and short spikes where DNA enters the slits but withdraws. DNA translocation events exhibit three distinct phases in their current-blockade traces—loading, translation, and exit. Coarse-grained molecular dynamics simulation allows the different polymer configurations of these phases to be identified. DNA molecules, including folds and knots in their polymer structure, are observed to slide through the slits with near-uniform velocity without noticeable frictional interactions of DNA with the confining graphene surfaces. It is anticipated that this new class of 2D-nanoslit devices will provide unique ways to study polymer physics and enable lab-on-a-chip biotechnology.

We have developed a fabrication methodology for label-free optical trapping of individual nanobeads and proteins in inverted-bowtie-shaped plasmonic gold nanopores. Arrays of these nanoapertures can be reliably produced using focused ion beam (FIB) milling with gap sizes of 10–20 nm, single-nanometer variation, and with a remarkable stability that allows for repeated use. We employ an optical readout where the presence of the protein entering the trap is marked by an increase in the transmission of light through the nanoaperture from the shift of the plasmonic resonance. In addition, the optical trapping force of the plasmonic nanopores allows 20-nm polystyrene beads and proteins, such as beta-amylase and Heat Shock Protein (HSP90), to be trapped for very long times (approximately minutes). On demand, we can release the trapped molecule for another protein to be interrogated. Our work opens up new routes to acquire information on the conformation and dynamics of individual proteins.

The dielectric breakdown approach for forming nanopores has greatly accelerated the pace of research in solid-state nanopore sensing, enabling inexpensive formation of nanopores via a bench top setup. Here the potential of tip-controlled local breakdown (TCLB) to fabricate pores 100× faster, with high scalability and nanometer positioning precision using an atomic force microscope (AFM) is demonstrated. A conductive AFM tip is brought into contact with a silicon nitride membrane positioned above an electrolyte reservoir. Application of a voltage pulse at the tip leads to the formation of a single nanoscale pore. Pores are formed precisely at the tip position with a complete suppression of multiple pore formation. In addition, the approach greatly accelerates the electric breakdown process, leading to an average pore fabrication time on the order of 10 ms, at least two orders of magnitude shorter than achieved by classic dielectric breakdown approaches. With this fast pore writing speed over 300 pores can be fabricated in half an hour on the same membrane.

Solid-state nanopores have emerged as promising platforms for biosensing including diagnostics for disease detection. Here we show nanopore experiments that detect CRISPR-dCas9, a sequence-specific RNA-guided protein system that specifically binds to a target DNA sequence. While CRISPR-Cas9 is acclaimed for its gene editing potential, the CRISPR-dCas9 variant employed here does not cut DNA but instead remains tightly bound at a user-defined binding site, thus providing an excellent target for biosensing. In our nanopore experiments, we observe the CRISPR-dCas9 proteins as local spikes that appear on top of the ionic current blockade signal of DNA molecules that translocate through the nanopore. The proteins exhibit a pronounced blockade signal that allows for facile identification of the targeted sequence. Even at the high salt conditions (1 M LiCl) required for nanopore experiments, dCas9 proteins are found to remain stably bound. The binding position of the target sequence can be read from the spike position along the DNA signal. We anticipate applications of this nanopore-based CRISPR-dCas9 biosensing approach in DNA-typing based diagnostics such as quick disease-strain identification, antibiotic-resistance detection, and genome typing.

The ability to control the motion of single biomolecules is key to improving a wide range of biophysical and diagnostic applications. Solid-state nanopores are a promising tool capable of solving this task. However, molecular control and the possibility of slow readouts of long polymer molecules are still limited due to fast analyte transport and low signal-to-noise ratios. Here, we report on a novel approach of actively controlling analyte transport by using a double-nanopore architecture where two nanopores are separated by only a ∼ 20 nm gap. The nanopores can be addressed individually, allowing for two unique modes of operation: (i) pore-to-pore transfer, which can be controlled at near 100% efficiency, and (ii) DNA molecules bridging between the two nanopores, which enables detection with an enhanced temporal resolution (e.g., an increase of more than 2 orders of magnitude in the dwell time) without compromising the signal quality. The simplicity of fabrication and operation of the double-barrel architecture opens a wide range of applications for high-resolution readout of biological molecules.

We report a simple and scalable technique for the fabrication of nanopore arrays on freestanding SiN and graphene membranes based on electron-beam lithography and reactive ion etching. By controlling the dose of the single-shot electron-beam exposure, circular nanopores of any size down to 16 nm in diameter can be fabricated in both materials at high accuracy and precision. We demonstrate the sensing capabilities of these nanopores by translocating dsDNA through pores fabricated using this method, and find signal-to-noise characteristics on par with transmission-electron-microscope-drilled nanopores. This versatile lithography-based approach allows for the high-throughput manufacturing of nanopores and can in principle be used on any substrate, in particular membranes made out of transferable two-dimensional materials.

We developed a method of precise isotope labeling to visualize the continuous growth of graphene by chemical vapour deposition (CVD). This method allows us to observe, as a function of time, the growth of graphene monocrystals at a resolution of a few seconds. This technique is used to extract the anisotropic growth rates, as well as investigate the formation of dendrites, and the dependence of growth rate on adsorption area of methane on copper. This technique also gives precise start times for individual graphene nucleation sites. We obtain a physical picture of the growth dynamics of graphene and its dependence on various parameters. Finally, our method is relevant to other CVD grown materials.