Biology subjects

Dekker, C.

Publications and source records attributed to Dekker, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Intercalator effects on DNA loop extrusion by SMC proteins

Intercalating dyes are widely used to visualize DNA in single-molecule experiments, but their effects on DNA-processing proteins remain incompletely characterized. Here, we study the effects of intercalators on DNA loop extrusion by SMC complexes. Intercalating dyes become fluorescent upon DNA binding, enabling direct visualization of extruded DNA loops. As dye intercalation alters the biophysical properties of DNA by lengthening and unwinding it, we examine how that modifies loop extrusion. We systematically characterize the effects of intercalators, and find that they stiffen DNA, partly neutralize its charge, and can substantially reduce the binding and ATPase activity of SMCs. We assess four dyes and conclude that SYBR Gold and SYTOX Orange are less invasive intercalators than SYTOX Green or YOYO-1. While loop extrusion is qualitatively not affected by intercalation, quantitative values for SMC binding and looping rates differ significantly from those in the absence of intercalators. We present a new dye-free loop-extrusion assay (utilizing local fluorescent spots along DNA), and find that loop extrusion proceeds at 1.75{+/-}0.26 kb/s for S. cerevisiae condensin without dyes, while adding intercalators leads to a two-fold lower rate. Our findings establish that intercalators quantitatively modify loop extrusion parameters, and we offer practical recommendations for minimizing their impact.

biophysics↗

bioRxiv2026

A modular DNA toolkit for single-molecule biophysics

Long, customizable DNA molecules are an essential tool for in vitro single-molecule biophysics. Most studies still rely on the {lambda}-phage genome, which has a fixed sequence, a non-uniform GC distribution, and limited options for modification. To overcome these limitations, we developed a modular DNA (ModDNA) toolkit that integrates in silico sequence design with cloning, to create long (up to 100 kb) DNA molecules with entirely user-defined sequences. We used a computational workflow with curated databases to design a motif-depleted sequence backbone, into which any desired sequence can be inserted at any position. Golden Gate assembly then builds these sequences into constructs, which we extend iteratively in defined increments up to 100 kb. We show precise placement of local features, e.g. a fluorophore, a bound protein, and a DNA-origami structure, and illustrate the construct's utility in single-molecule visualization assays, including DNA loop extrusion by human condensin I. ModDNA offers a scalable, sequence-programmable alternative to {lambda}-DNA and other restrictive substrates. More broadly, the motif-aware feature-free design connects single-molecule biophysics to emerging efforts in machine-guided regulatory-sequence design and synthetic-genome engineering, where controlled programmable sequence context is increasingly essential.

biophysics↗

bioRxiv2026

Dilatable DNA origami nanopores as nuclear pore mimics

The nuclear pore complex (NPC) is a massive protein system that controls all nucleocytoplasmic transport via a dynamic network of intrinsically disordered proteins called FG-Nups. We developed an NPC-mimicking DNA origami nanostructure that self-assembles into a tetrameric, octagonal nanopore with an inner diameter that, through addition of DNA oligonucleotides, can be tuned in a user-defined fashion from 57 to 66 nm - mimicking the contracted and dilated states of NPCs. We demonstrated that these origami nanopores can dilate within minutes and recontract at slower timescales. Both contracted and dilated pores can be inserted into lipid bilayers, where they maintain their conformation and allow transmembrane transport of fluorescent proteins. With outer diameters up to 87 nm, these are, to our knowledge, the largest DNA origami nanopores inserted into lipid membranes. Functionalization of the pores with the FG-Nup Nsp1 reduces non-specific cargo diffusion across the lipid membrane while facilitating translocation of the transport receptor Kap95, demonstrating transport selectivity much like biological NPCs. The work establishes a size-tunable platform to dissect nuclear transport mechanisms, with potential applications for engineering programmable artificial channels.

biophysics↗

bioRxiv2026

Division of Synthetic Cells Using a Genomically Encoded One-Protein Divisome

Synthetic biology is emerging as a powerful tool to understand life by building minimal synthetic cells whose functions are encoded within their genome. An essential function of cells is abscission, the final step of cell division that breaks the last connection between two emerging daughter cells. Abscission is challenging to reconstitute as it is energetically unfavorable to rearrange the cell membrane. Here, we demonstrate a minimal one-component abscission machinery for synthetic cells using the bacterial protein dynamin A (DynA), which is uniquely capable of driving scission inside membrane necks. Cell-free expression of this synthetic cell module is complicated by the large size of DynA (137 kDa) and the presence of a hydrophobic lipid-binding loop within its structure. We overcome these challenges by expressing DynA inside lipid vesicles with a high fraction of negatively charged lipids. We find that DynA enriches at the bridges of dumbbell-shaped vesicles, where it drives membrane scission and full division. The ability to monitor DynA dynamics through genomic encoding further provides hints about the mechanism of abscission including cooperative enrichment at the necks and constriction of the necks. Finally, we demonstrate that the smaller (71 kDa) dynamin D1 domain alone exerts the same function, which is beneficial for future integration with other synthetic cell modules since its much smaller gene size reduces the burden on the synthetic genome. This autonomously operating abscission machinery presents the first example of a self-dividing synthetic cell, marking an important step towards constructing a synthetic cell that is able to sustainably replicate.

synthetic biology↗

bioRxiv2026

Charge and steric hindrance in the glycocalyx govern membrane interactions

The outer leaflet of the plasma membrane - the cells interface with exogenous biomolecules and particles - is largely electrostatically neutral and highly viscous, conditions that are generally unfavorable for fusion of external membrane-bound objects such as liposomes. Yet living cells efficiently interact and fuse with charged liposomes, revealing a discrepancy between synthetic and biological membranes. Here, we identify the glycocalyx, the carbohydrate-rich layer that coats cells, as the component that explains this mismatch. Using synthetic membranes reconstituted with an artificial glycocalyx, we resolve how electrostatics and steric hindrance regulate the interactions of cationic liposomes with synthetic membranes. Charged glycolipids are found to be sufficient to convert non-fusogenic neutral membranes into fusion-competent membranes. In contrast, large polymers in the glycocalyx suppress fusion. Steric hindrance acts primarily at the docking stage, while fusion proceeds through a long-lived hemifusion intermediate. Strikingly, these large differences occur without measurable changes in membrane viscosity or phase state, demonstrating that glycocalyx-mediated control is independent of the membrane mechanics. Selected glycocalyx components can also promote endocytic budding and cargo internalization. We thus show that the glycocalyx is a tunable physicochemical regulator of liposome docking, fusion, and internalization. The findings provide a framework for understanding how biological agents and therapeutics engage cell membranes and they offer key design principles for synthetic cells.

biophysics↗

bioRxiv2026

Lipid-conjugated DNA enables on-demand delivery of lipids and proteins to synthetic cells

The bottom-up construction of synthetic cells based on giant unilamellar vesicles (GUVs) is a central goal in synthetic biology. Achieving targeted changes in membrane and cytoplasmic composition with temporal control remains challenging however. DNA-mediated fusion with small vesicles ([~]100 nm large unilamellar vesicles; LUVs) has been proposed as a strategy to deliver lipids and cytosolic contents in a programmable manner. However, in vitro, membrane fusion is generally found to be inefficient and poorly controllable for reasons that are poorly understood. Here, we present an approach based on lipid-conjugated DNA (LiNA) to mediate programmable fusion between LUVs and micron-sized GUVs, which we quantitatively monitor with confocal microscopy at the single-GUV level. We show that lipid and content mixing both occur with high efficiency over a wide range of LiNA concentrations, demonstrating that LiNAs indeed induce robust membrane fusion. Furthermore, we show that LiNA-mediated fusion provides a powerful tool to deliver cytosolic biomolecules, enabling control over internal activities. Our findings establish a quantitative framework for studying fusion-driven processes in synthetic cells and provide a versatile platform for the programmable delivery of lipids and cytosolic cargoes - thus advancing the development of synthetic cells that can grow and adapt through fusion-based uptake of molecular building blocks.

synthetic biology↗

bioRxiv2025

Terminal conjugation enables nanopore sequencing of peptides

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.

biophysics↗

bioRxiv2025

Spatial control of karyopherin binding avidity within NPC mimics revealed by designer FG-Nucleoporins

Nucleocytoplasmic transport occurs via nuclear pore complexes (NPCs), [~]40-60 nm wide pores lined with intrinsically disordered proteins that are rich in Phe-Gly motifs (FG-Nups) that form a selective barrier. Molecules larger than [~]50 kDa are increasingly blocked for transport unless they are bound to a nuclear transport receptor (NTR). How the amino acid sequence of FG-Nups contribute to this is not fully understood. Here, we present de novo designed artificial FG-Nups with a systematically varied FG-repeat spacing and charge-to-hydrophobicity ratio (C/H). Starting from a reference sequence termed NupY (with the average properties of natural yeast GLFG- Nups), we designed, synthesized, and experimentally tested a library of NupY variants using QCM-D experiments and phase separation assays. We find that the spacing between FG-motifs governs Kap95 absorption into the FG- Nup phase, while increasing C/H results in higher avidity for Kap95 due to an increased accessibility of FG-motifs. Molecular dynamics simulations of transport through NupY-coated pores show a reduced barrier function for noncohesive high-C/H-ratio variants and the highest transport selectivity for designs close to native GLFG-Nups. We postulate that a balance between entropic repulsion and enthalpic gain from multivalent Kap-FG-Nup interactions drives the spatial and temporal partitioning of Kaps in the NPC.

biophysics↗

bioRxiv2025

Lipid flip flop regulates the shape of growing and dividing synthetic cells

Cells grow their boundaries by incorporating newly synthesized lipids into their membranes as well as through fusion of intracellular vesicles. As these processes yield trans-bilayer imbalances in lipid numbers, cells must actively flip lipid molecules across the bilayer to enable growth. Using giant and small unilamellar vesicles (GUVs and SUVs, respectively), we here recapitulate cellular growth and division under various conditions of transmembrane flip flop of lipids. By dynamically monitoring the changes in reduced volume and spontaneous curvature of GUVs that grow by fusion of many small SUVs, the morphology of these growing synthetic cells is quantified. We demonstrate that lipid flip flop relaxes curvature stresses and yields more symmetrically sized buds. Further increasing the neck curvature is shown to lead to bud scission. The mechanisms presented here offer fundamental insights into cell growth and division, which are important for understanding early protocells and designing synthetic cells that are able to grow and divide.

synthetic biology↗

bioRxiv2025

Two CTCF motifs impede cohesin-mediated DNA loop extrusion

Cohesin extrudes DNA into loops and is positioned along the genome by stalling at 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, that hinder LE. KTYQR is found to fully impede LE activity, while YDF hinders cohesin to complete 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. One-Sentence SummaryThe N-terminus of CTCF employs two independent motifs that synergistically stall cohesin-mediated DNA loop extrusion.

biophysics↗

bioRxiv2024

A microfluidic platform for extraction and analysis of bacterial genomic DNA

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.

biophysics↗

bioRxiv2024

Interaction hierarchy among Cdv proteins drives recruitment to membrane necks

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 can bind to lipid membranes, but does so more efficiently when it is 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.

biochemistry↗

bioRxiv2024

DNA supercoiling enhances DNA condensation by ParB proteins

The ParABS system plays a critical role in bacterial chromosome segregation. The key component of this system, ParB, loads and spreads along DNA to form a local protein-DNA condensate known as a partition complex. As bacterial chromosomes are heavily supercoiled due to the continuous action of RNA polymerases, gyrases, and nucleoid-associated proteins, it is important to study the impact of DNA supercoiling on the ParB-DNA partition complex formation. Here, we use an in vitro single- molecule assay to visualize ParB on supercoiled DNA. Unlike most DNA-binding proteins, individual ParB proteins are found to not pin plectonemes on supercoiled DNA, but freely diffuse along supercoiled DNA. We find that DNA supercoiling enhances ParB-DNA condensation which initiates at lower ParB concentrations than on DNA that is torsionally relaxed. ParB proteins induce a DNA- protein condensate that strikingly absorbs all supercoiling writhe. Our findings provide mechanistic insights that have important implications for our understanding of bacterial chromosome organization and segregation.

biophysics↗

bioRxiv2024

Resolving sulfation PTMs on a plant peptide hormone using nanopore sequencing

Peptide phytohormones are decorated with post-translational modifications (PTMs) that are crucial for receptor recognition. Tyrosine sulfation on these hormones is essential for plant growth and development1. Measuring the occurrence and position of sulfotyrosine is, however, compromised by major technical challenges during isolation and detection2. We recently introduced a nanopore peptide sequencing method that sensitively detects PTMs at the single-molecule level3. By translocating PTM variants of the plant pentapeptide hormone phytosulfokine (PSK) through a nanopore, we here demonstrate 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 new tool for identifying sulfation on peptide phytohormones and promising wider applications to identify protein PTMs.

biophysics↗

bioRxiv2024

Compliant DNA Origami Nanoactuators as Size-Selective Nanopores

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, we harness the unique combined properties of DNA origami nanotechnology, machine-inspired design, and synthetic biology, 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 three 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 behaviour 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.

biophysics↗

bioRxiv2024

All eukaryotic SMC proteins induce a twist of -0.6 at each DNA-loop-extrusion step

Eukaryotes carry three types of Structural Maintenance of Chromosomes (SMC) protein complexes, condensin, cohesin, and SMC5/6, which are ATP-dependent motor proteins that remodel the genome via DNA loop extrusion. SMCs modulate DNA supercoiling, but it has remained incompletely understood how this is achieved. Here we present a single-molecule magnetic tweezers assay that directly measures how much twist is induced by an individual SMC in each loop-extrusion step. We demonstrate that all three SMC complexes induce the same large negative twist (i.e., a linking number change {Delta}Lk of -0.6 at each loop-extrusion step) into the extruded loop, independent of step size. Using ATP-hydrolysis mutants and non-hydrolysable ATP analogues, we find that ATP binding is the twist-inducing event during the ATPase cycle, which coincides with the force-generating loop-extrusion step. The fact that all three eukaryotic SMC proteins induce the same amount of twist indicates a common DNA-loop-extrusion mechanism among these SMC complexes.

biophysics↗

bioRxiv2024

Cohesin supercoils DNA during loop extrusion

Cohesin extrudes genomic DNA into loops that promote chromatin assembly, gene regulation and recombination. Here we show that cohesin introduces negative supercoils into extruded DNA. Supercoiling requires engagement of cohesins ATPase heads, DNA clamping by these heads, and a DNA binding site on cohesins hinge, indicating that cohesin supercoils DNA when constraining it between the hinge and the clamp. Our results suggest that DNA extrusion stops once cohesin reaches its stall torque during supercoiling, and a cohesin mutant predicted to stall at lower torque forms shorter loops in cells. These results indicate that supercoiling is an integral part of the loop extrusion mechanism and that cohesin controls genome architecture not only by looping DNA but also by supercoiling it.

biochemistry↗

bioRxiv2023

SMC motor proteins extrude DNA asymmetrically and contain a direction switch

Structural Maintenance of Chromosomes (SMC) complexes organize the genome via DNA loop extrusion. While 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, while 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. STAG1 stabilizes NIPBL on cohesin, preventing NIPBL turnover and direction switches. The findings reveal that SMCs, surprisingly, contain a direction switch subunit. HighlightsO_LIAll eukaryotic SMC complexes extrude DNA asymmetrically. C_LIO_LIApparent symmetric loop extrusion is the result of frequent direction switches. C_LIO_LIn human cohesin, loop-extrusion direction changes require exchange of NIPBL. C_LIO_LISTAG1 stabilizes NIPBL on human cohesin. C_LI

molecular biology↗