Nanopore sequencing of peptides holds great promise for single-molecule proteomics, but robust conjugation strategies to adapt native peptides for motor-enzyme-driven translocation have yet to be developed. Here, we establish terminally directed DNA-peptide conjugation chemistry strategies that expand the applicability of nanopore sequencing beyond synthetic model systems to natural peptides. At the N terminus, omniligase catalyzes rapid and peptide ligation of a DNA handle under mild conditions. At the C terminus, photoredox decarboxylative ligation introduces a bioorthogonal linker that enables CuAAC-mediated DNA attachment that ensures proper stretching and translocation of short peptides through the nanopore. Our study reveals that long peptides can be sequenced with single-end conjugation, while short or neutral peptides require threading tails. Positively charged peptides cannot be translocated under the same electric field but can be sequenced after charge neutralization. The data demonstrate controlled nanopore readouts of peptides that differ widely in length, charge, and sequence. This framework establishes a versatile chemical foundation for adapting natural peptides to nanopore sequencing, advancing single-molecule proteomic analysis.

Synthetic cells (SynCells) are artificial constructs designed to mimic cellular functions, offering insights into fundamental biology, as well as promising impact in the fields of medicine, biotechnology, and bioengineering. Achieving a functional SynCell from the bottom up, i.e. by assembling it from molecular components, requires a global collaboration to overcome the many challenges of engineering and assembling life-like modules while addressing biosafety, equity, and ethical concerns in order to guide responsible innovation. Here, we highlight major scientific hurdles, such as the integration of functional modules by ensuring compatibility across diverse synthetic subsystems, and we propose strategies to advance the field.

Due to its pivotal role as a regulator of nucleocytoplasmic transport, the structure and dynamic gating mechanism of the nuclear pore complex (NPC) is a subject of immense interest. Here, we report key recent advancements discussed at the Selective Transport Control in Biological and Biomimetic Nanopores meeting (Monte Verità, Switzerland, 2024) that gathered NPC experts from a range of disciplines. Novel insights were reported from cutting-edge super-resolution techniques that enable the direct interrogation of the NPC’s dynamic central transporter; computational models that unravel the mechanisms of the selective barrier; and synthetic NPC mimics as valuable in vitro models for delineating NPC permeability and transport dynamics. Altogether, three major insights were highlighted: (i) the presence of dynamically organised nuclear transport pathways within the NPC, (ii) the role of nuclear transport receptors that enrich and reinforce the NPC’s selective permeability barrier, and (iii) the ability of DNA origami nanostructures to mimic aspects of the NPC with unprecedented precision. Overall, the advancements marked a convergence in our understanding of NPC function by unraveling its dynamic gating mechanism at the nanoscale.

Synchronization plays a crucial role in the dynamics of living organisms. Uncovering the mechanism behind it requires an understanding of individual biological oscillators and the coupling forces between them. Here, a single-cell assay is developed that studies rhythmic behavior in the motility of E. coli cells that can be mutually synchronized. Circular microcavities are used to isolate E. coli cells that swim along the cavity wall, resulting in self-sustained oscillations. Connecting these cavities by microchannels yields synchronization patterns with phase slips. It is demonstrated that the coordinated movement observed in coupled E. coli oscillators follows mathematical rules of synchronization which is used to quantify the coupling strength. These findings advance the understanding of motility in confinement, and open up new opportunities for engineering networks of coupled oscillators in microbial active matter.

DNA loop extrusion by SMC proteins is a key process underlying chromosomal organization. It is unknown how loop extruders interact with telomeres where DNA is densely covered with proteins. Using complementary in vivo and in vitro single-molecule approaches, we study how loop-extruding condensin interacts with Rap1, the telomeric DNA-binding protein of Saccharomyces cerevisiae. We show that dense linear Rap1 arrays can completely halt DNA loop extrusion, with a blocking efficiency depending on the array length and the DNA gap size between proteins. In anaphase cells, dense Rap1 arrays are found to accumulate condensin and to cause a local chromatin decompaction, as monitored with a microscopy-based approach, with direct implications for the resolution of dicentric chromosomes produced by telomere fusions. Our findings show that linear arrays of DNA-bound proteins can efficiently halt DNA loop extrusion by SMC proteins, which may impact cellular processes from telomere functions to transcription and DNA repair.

Human cohesin extrudes DNA into loops and is positioned along the genome by stalling at the human CCCTC-binding factor (CTCF) upon encountering its N-terminal region (NTR). The mechanism underlying this stalling, however, is unresolved. Using single-molecule assays that monitor DNA loop extrusion (LE) in the presence of NTR fragments, we identify two amino acid motifs, YDF and KTYQR, which hinder LE. KTYQR is found to completely block LE activity, while YDF hinders cohesin from completing LE step cycles and converts cohesin into a unidirectional extruder by strengthening the affinity of STAG1 to DNA. We thus identify two distinct NTR motifs that stall LE via different yet synergistic mechanisms, highlighting the multifaceted ways employed by CTCF to modulate LE to shape and regulate genomes.

Structural maintenance of chromosomes (SMC) complexes organize the genome via DNA loop extrusion. Although some SMCs were reported to do so symmetrically, reeling DNA from both sides into the extruded DNA loop simultaneously, others perform loop extrusion asymmetrically toward one direction only. The mechanism underlying this variability remains unclear. Here, we examine the directionality of DNA loop extrusion by SMCs using in vitro single-molecule experiments. We find that cohesin and SMC5/6 do not reel in DNA from both sides, as reported before, but instead extrude DNA asymmetrically, although the direction can switch over time. Asymmetric DNA loop extrusion thus is the shared mechanism across all eukaryotic SMC complexes. For cohesin, direction switches strongly correlate with the turnover of the subunit NIPBL, during which DNA strand switching may occur. Apart from expanding by extrusion, loops frequently diffuse and shrink. The findings reveal that SMCs, surprisingly, can switch directions.

Cohesin extrudes genomic DNA into loops that promote chromatin assembly, gene regulation, and gene recombination. Loop extrusion depends on large-scale conformational changes in cohesin, but how these translocate DNA is poorly understood. Here, we provide evidence that cohesin negatively supercoils DNA during loop extrusion. Supercoiling requires the engagement of cohesin’s ATPase heads, DNA clamping by these heads, and a DNA-binding site on cohesin’s hinge, indicating that cohesin twists DNA when constraining it between the hinge and the clamp. A cohesin mutant defective in negative supercoiling forms shorter loops in cells, and a similar, although weaker, phenotype is observed after the depletion of topoisomerase I. These results suggest that supercoiling is an integral part of the loop-extrusion mechanism and that relaxation of supercoiled DNA is required for cohesin-mediated loop extrusion and genome architecture.

In September 2023, the Biology and Physics of Prokaryotic Chromosomes meeting ran at the Lorentz Center in Leiden, The Netherlands. As part of the workshop, those in attendance developed a series of discussion points centered around current challenges for the field, how these might be addressed, and how the field is likely to develop over the next 10 years. The Lorentz Center staff facilitated these discussions via tools aimed at optimizing productive interactions. This Perspective article is a summary of these discussions and reflects the state-of-the-art of the field. It is expected to be of help to colleagues in advancing their own research related to prokaryotic chromosomes and inspiring novel interdisciplinary collaborations. This forward-looking perspective highlights the open questions driving current research and builds on the impressive recent progress in these areas as represented by the accompanying reviews, perspectives, and research articles in this issue. These articles underline the multi-disciplinary nature of the field, the multiple length scales at which chromatin is studied in vitro and in and highlight the differences and similarities of bacterial and archaeal chromatin and chromatin-associated processes.

Bacterial cells organize their genomes into a compact hierarchical structure called the nucleoid. Studying the nucleoid in cells faces challenges because of the cellular complexity while in vitro assays have difficulty in handling the fragile megabase-scale DNA biopolymers that make up bacterial genomes. Here, we introduce a method that overcomes these limitations as we develop and use a microfluidic device for the sequential extraction, purification, and analysis of bacterial nucleoids in individual microchambers. Our approach avoids any transfer or pipetting of the fragile megabase-size genomes and thereby prevents their fragmentation. We show how the microfluidic system can be used to extract and analyze single chromosomes from B. subtilis cells. Upon on-chip lysis, the bacterial genome expands in size and DNA-binding proteins are flushed away. Subsequently, exogeneous proteins can be added to the trapped DNA via diffusion. We envision that integrated microfluidic platforms will become an essential tool for the bottom-up assembly of complex biomolecular systems such as artificial chromosomes.

Cell division in the crenarchaea is accomplished by the Cdv system. In Sulfolobus cells, it was observed that an initial non-contractile ring of CdvA and CdvB forms at the mid location of the cell, which is followed by a second ring of CdvB1 and CdvB2 that appear to drive the constriction of the cell membrane. Here, we use an in vitro reconstituted system to explore how protein interactions among these Cdv proteins govern their recruitment to the membrane. We show that CdvA does not bind the membrane unless in complex with CdvB. We find that CdvB2 can polymerize if its self-inhibitory domain is removed, and that by itself is exhibits poor binding to the membrane. However, CdvB2 can be efficiently recruited to the membrane by both CdvB1 and CdvB. Furthermore, the CdvB1:CdvB2 co-polymer can be recruited to the membrane by CdvA:CdvB. By reconstituting these proteins in dumbbell-shaped liposomes, we show that Cdv proteins have a strong preference to localize at membrane necks of high curvature. Our findings clarify many of the mutual protein interactions of the Cdv system and their interaction with the membrane, thus helping to build a mechanistic understanding of cell division in archaeal cells.

Transcription-coupled supercoiling of DNA is a key factor in chromosome compaction and the regulation of genetic processes in all domains of life. It has become common knowledge that, during transcription, the DNA-dependent RNA polymerase (RNAP) induces positive supercoiling ahead of it (downstream) and negative supercoils in its wake (upstream), as rotation of RNAP around the DNA axis upon tracking its helical groove gets constrained due to drag on its RNA transcript. Here, we experimentally validate this so-called twin-supercoiled-domain model with in vitro real-time visualization at the single-molecule scale. Upon binding to the promoter site on a supercoiled DNA molecule, RNAP merges all DNA supercoils into one large pinned plectoneme with RNAP residing at its apex. Transcription by RNAP in real time demonstrates that up- and downstream supercoils are generated simultaneously and in equal portions, in agreement with the twin-supercoiled-domain model. Experiments carried out in the presence of RNases A and H, revealed that an additional viscous drag of the RNA transcript is not necessary for the RNAP to induce supercoils. The latter results contrast the current consensus and simulations on the origin of the twin-supercoiled domains, pointing at an additional mechanistic cause underlying supercoil generation by RNAP in transcription.

Bacterial chromosomes are folded into tightly regulated three-dimensional structures to ensure proper transcription, replication, and segregation of the genetic information. Direct visualization of chromosomal shape within bacterial cells is hampered by cell-wall confinement and the optical diffraction limit. Here, we combine cell-shape manipulation strategies, high-resolution fluorescence microscopy techniques, and genetic engineering to visualize the shape of unconfined bacterial chromosome in real-time in live Bacillus subtilis cells that are expanded in volume. We show that the chromosomes predominantly exhibit crescent shapes with a non-uniform DNA density that is increased near the origin of replication (oriC). Additionally, we localized ParB and BsSMC proteins – the key drivers of chromosomal organization – along the contour of the crescent chromosome, showing the highest density near oriC. Opening of the BsSMC ring complex disrupted the crescent chromosome shape and instead yielded a torus shape. These findings help to understand the threedimensional organization of the chromosome and the main protein complexes that underlie its structure.

Cytoskeletal protein filaments such as actin and microtubules confer mechanical support to cells and facilitate many cellular functions such as motility and division. Recent years have witnessed the development of a variety of molecular scaffolds that mimic such filaments. Indeed, filaments that are programmable and compatible with biological systems may prove useful in studying or substituting such proteins. Here, we explore the use of ssRNA tiles to build and modify filaments in vitro. We engineer a number of functionalities that are crucial to the function of natural proteins filaments into the ssRNA tiles, including the abilities to assemble or disassemble filaments, to tune the filament stiffness, to induce membrane binding, and to bind proteins. This work paves the way for building dynamic cytoskeleton-mimicking systems made out of rationally designed ssRNA tiles that can be transcribed in natural or synthetic cells.

Biological nanopores crucially control the import and export of biomolecules across lipid membranes in cells. They have found widespread use in biophysics and biotechnology, where their typically narrow, fixed diameters enable selective transport of ions and small molecules, as well as DNA and peptides for sequencing applications. Yet, due to their small channel sizes, they preclude the passage of large macromolecules, e.g., therapeutics. Here, the unique combined properties of DNA origami nanotechnology, machine-inspired design, and synthetic biology are harnessed, to present a structurally reconfigurable DNA origami MechanoPore (MP) that features a lumen that is tuneable in size through molecular triggers. Controllable switching of MPs between 3 stable states is confirmed by 3D-DNA-PAINT super-resolution imaging and through dye-influx assays, after reconstitution of the large MPs in the membrane of liposomes via an inverted-emulsion cDICE technique. Confocal imaging of transmembrane transport shows size-selective behavior with adjustable thresholds. Importantly, the conformational changes are fully reversible, attesting to the robust mechanical switching that overcomes pressure from the surrounding lipid molecules. These MPs advance nanopore technology, offering functional nanostructures that can be tuned on-demand – thereby impacting fields as diverse as drug delivery, biomolecule sorting, and sensing, as well as bottom-up synthetic biology.

Bacterial cells require DNA segregation machinery to properly distribute a genome to both daughter cells upon division. The most common system involved in chromosome and plasmid segregation in bacteria is the ParABS system. A core protein of this system - partition protein B (ParB) - regulates chromosome organization and chromosome segregation during the bacterial cell cycle. Over the past decades, research has greatly advanced our knowledge of the ParABS system. However, many intricate details of the mechanism of ParB proteins were only recently uncovered using in vitro single-molecule techniques. These approaches allowed the exploration of ParB proteins in precisely controlled environments, free from the complexities of the cellular milieu. This review covers the early developments of this field but emphasizes recent advances in our knowledge of the mechanistic understanding of ParB proteins as revealed by in vitro single-molecule methods. Furthermore, we provide an outlook on future endeavors in investigating ParB, ParB-like proteins, and their interaction partners.

Eukaryotes carry three types of structural maintenance of chromosome (SMC) protein complexes, condensin, cohesin, and SMC5/6, which are ATP-dependent motor proteins that remodel the genome via DNA loop extrusion (LE). SMCs modulate DNA supercoiling but remains incompletely understood how this is achieved. Using a single-molecule magnetic tweezers assay that directly measures how much twist is induced by individual SMCs in each LE step, we demonstrate that all three SMC complexes induce the same large negative twist (i.e., linking number change ΔL _k of ~−0.6 at each LE step) into the extruded loop, independent of step size and DNA tension. Using ATP hydrolysis mutants and nonhydrolyzable ATP analogs, we find that ATP binding is the twist-inducing event during the ATPase cycle, coinciding with the force-generating LE step. The fact that all three eukaryotic SMC proteins induce the same amount of twist indicates a common DNA-LE mechanism among these SMC complexes.

Protein sequencing and the identification of post-translational modifications are key to understanding cellular signalling pathways and metabolic processes in health and disease. Nanopores, that is, nanometre-sized holes in a membrane, were previously put to use for DNA and RNA sequencing, offering single-molecule sensitivity and long read lengths. This prompted efforts to engineer nanopores for the high-throughput sequencing of peptides and proteins. In this Review, we discuss research towards single-molecule protein sequencing using biological nanopores, investigating how their sensitivity allows the discrimination of all 20 amino acids. We outline how fingerprinting of proteins is facilitated by using motor proteins and electro-osmotic flow to promote the slow translocation of proteins through nanopores. Moreover, we examine applications of nanopores to the identification of post-translational modifications, highlighting the potential of this technology for fundamental and clinical proteomic studies. Finally, we outline the advantages and limitations of nanopore systems for protein sequencing and the challenges that remain to be overcome for realizing de novo nanopore protein sequencing.

Graphene-drum-enabled nanomotion detection can play an important role in probing life at the nanoscale. By combining micro- and nanomechanical systems with optics, nanomotion sensors bridge the gap between mechanics and cellular biophysics. They have allowed investigation of processes involved in metabolism, growth, and structural organization of a large variety of microorganisms, ranging from yeasts to bacterial cells. Using graphene drums, these processes can now be resolved at the single-cell level. In this Perspective, we discuss the key achievements of nanomotion spectroscopy and peek forward into the prospects for application of this single-cell technology in clinical settings. Furthermore, we discuss the steps required for implementation and look into applications beyond microbial sensing.

Peptide hormones are decorated with post-translational modifications (PTMs) that are crucial for receptor recognition. Tyrosine sulfation on plant peptide hormones is, for example, essential for plant growth and development. Measuring the occurrence and position of sulfotyrosine is, however, compromised by major technical challenges during isolation and detection. Nanopores can sensitively detect protein PTMs at the single-molecule level. By translocating PTM variants of the plant pentapeptide hormone phytosulfokine (PSK) through a nanopore, we here demonstrate the accurate identification of sulfation and phosphorylation on the two tyrosine residues of PSK. Sulfation can be clearly detected and distinguished (>90%) from phosphorylation on the same residue. Moreover, the presence or absence of PTMs on the two close-by tyrosine residues can be accurately determined (>96% accuracy). Our findings demonstrate the extraordinary sensitivity of nanopore protein measurements, providing a powerful tool for identifying position-specific sulfation on peptide hormones and promising wider applications to identify protein PTMs.