Biology subjects

Aksimentiev, A.

Publications and source records attributed to Aksimentiev, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Expandable DNA-origami rings as a nanomechanical platform for studying disordered nucleoporins

Phe-Gly-rich nucleoporins (FG-Nups) within the nuclear pore complex (NPC) form a diffusion barrier that controls the exchange of macromolecules between the nucleus and cytoplasm. The behaviors of these intrinsically disordered FG-Nups are fundamental to the NPC function but remain difficult to probe within the dynamic, native nuclear pore. To bridge this knowledge gap, here we build expandable DNA-origami rings with tunable elasticity and study FG-Nups site-specifically tethered in such mechanically responsive NPC mimics. The elastic deformation of DNA rings, measured by single-particle TEM, reveals the type (attraction or repulsion) and strength of FG-Nup interactions that shape the collective morphology of these unstructured proteins. Analyzing homotypic interactions of five FG-Nup species finds diverse FG-Nup behaviors that can be classified as repulsive/extended (Nsp1), moderately cohesive/extended (Nup62, Nup153, and Nup214), and strongly cohesive/compact (Nup98). Furthermore, we examine how FG-Nups' primary sequence, O-glycosylation, grafting density, and binding to nuclear transport receptors (NTRs) impact their biophysical state. For example, O-GlcNAcylation reduced FG-Nup cohesiveness, switching Nup98 from a collapsed state to more extended conformations. Our work thus elucidates spatially confined Nup-Nup and Nup-NTR interactions within an NPC-like nanopore, suggests a previously underappreciated role of FG-Nups in modulating NPC dilation, and establishes a generalizable method for studying multivalent interactions among disordered proteins.

biophysics↗

bioRxiv2026

DNA: A Nanoscale Archimedes Screw

Rotating helices have been utilized for many purposes, including the transport of solid material and fluids within man-made machines, for a little over two millennia. Here, we show that the rotation of a biological helical molecule--a DNA duplex--can move water and ions through a nanoscale pore. While the rotation-induced flow of water is generated by the steric shape of the DNA molecule, an even faster transport of cations is caused by electrostatic interactions. The rotation-induced ion flux is found to depend on the cation type, offering potential utility for ion separation. Finally, we show that the torque-driven duplex can move ions against a concentration gradient, realizing the Archimedes screw principle at the nanoscale. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/734429v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@14e2829org.highwire.dtl.DTLVardef@2ebc37org.highwire.dtl.DTLVardef@46d16forg.highwire.dtl.DTLVardef@1600c19_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

Improving All-Atom Molecular Dynamics Models for Quantitative Prediction of Nanopore Blockade Current

All-atom molecular dynamics has become an indispensable tool in development of nanopore sensors of biological information. In a typical nanopore experiment, measurements of ionic current flowing through a nanopore report on the chemical structure of biomolecules that pass through the nanopore. Such experiments alone are often insufficient to relate the structure of the biomolecules to the ionic current modulations. The molecular dynamics method can establish such a relationship directly through a brute force simulation under applied electric field. Here, we examine the ability of molecular dynamics force fields to reproduce experimentally measured nanopore blockade currents produced by single-stranded DNA. Our simulations show that none of the "off the shelf" force fields (CHARMM36, AMBER Parmbsc1 and DES-AMBER) is capable of reproducing experimental data with the desired level of accuracy. To improve the accuracy, we examined and refined interactions between ions, protein nanopores and DNA, guided by experiments designed specifically for this purpose. Ultimately, the introduction of surgical corrections to non-bonded interactions within the CHARMM36 force field produced a favorable agreement between simulation and experiment. This refined parameterization, initially developed for nanopore sensing simulations, may have broader applications in computational studies of DNA-protein systems.

biophysics↗

bioRxiv2026

Solvent ordering in a fully packaged RNA bacteriophage and the mechanics of genome delivery

Self-assembly of an RNA virus requires a thermodynamic state stable enough to confine the highly charged genome yet primed for genome delivery. By simulating the behavior of every atom in a packaged bacteriophage, we determined the physical factors enabling such a state to emerge. We show that the electrical charge of the RNA genome is neutralized by both a cloud of mobile counterions and polarized water that exhibits a long-range order despite stochastic motion of the unstructured parts of the genome. Acting as an intermolecular glue, the solvent equipoises self-repulsion of the RNA genome with attraction of RNA to specific sites at the capsid, accommodating the mechanical stress produced by the capsids fluctuations. Simulations of genome extraction show that specific RNA-protein contacts can withstand extreme forces, supporting mechanical extraction as a mechanism of genome delivery. Together, our findings detail how self-assembled viruses balance the conflicting biophysical demands of confinement and infection.

biophysics↗

bioRxiv2026

DNA-Lipid Nanodiscs with a Polyethylene Glycol Interface

Nanoscale bilayer mimetics such as protein or polymer-based nanodiscs are versatile tools to study the physical chemistry of lipid bilayers or the structures and functions of membrane proteins. Here, we introduce DNA-Lipid Nanodiscs (DLNs) in which the interface between hydrophobic lipids and the charged DNA is mediated through amphiphilic poly(ethylene)glycol (PEG). For this, we modified oligonucleotides with PEG and hybridized them to a single-stranded ring to form functionalized minicircles with a well-defined diameter. The center of these minicircles can be filled with a lipid bilayer through addition of detergent-solubilized lipids followed by detergent removal. Simulations reveal that the methylene groups in PEG form dynamic interactions with the acyl chains of lipids, effectively shielding the hydrophobic mismatch. As proof of concept towards incorporation of complex membrane proteins, we inserted the biotinylated transmembrane domain of synaptobrevin into these nanodiscs and bound them to streptavidin-modified quantum dots as a marker for successful incorporation. We envision these atomically precise, modular DNA scaffolds to be widely applicable in future studies of membrane proteins and nanoscale lipid membranes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/705827v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@17cfcb5org.highwire.dtl.DTLVardef@b2dd2corg.highwire.dtl.DTLVardef@d6899aorg.highwire.dtl.DTLVardef@e400c4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Ultrafast CTCF dynamics control cohesin barrier function

Genomes are organized into chromatin loops through cohesin-mediated extrusion, with CTCF acting as a polar boundary element. As cohesin approaches CTCF at kilobase-per-second speeds, it must rapidly choose whether to stall or bypass. How CTCF encodes this probabilistic decision within a brief encounter window has remained unclear. Here we show that CTCF governs this probabilistic outcome by rapidly sampling a dynamic ensemble of conformations generated by spontaneous rearrangements of its DNA-binding zinc fingers. This ensemble is tuned by DNA sequence, CpG methylation, nearby nucleosomes, and the cohesin regulator PDS5A before cohesin engagement. Upon cohesin binding, PDS5A enhances loop-anchor mechanical stability, reinforcing orientation-dependent boundaries. These findings establish conformational ensemble tuning, rather than static occupancy, as a regulatory principle linking base pair-scale motions to megabase-scale genome organization. One sentence summaryChromatin boundary function is governed not by CTCF occupancy alone, but by a tunable ensemble of DNA-bound conformations that probabilistically gates cohesin capture.

biophysics↗

bioRxiv2024

Single molecule delivery into living cells

Controlled manipulation of cultured cells by delivery of exogenous macromolecules is a cornerstone of experimental biology. Here we describe a platform which uses nanopipettes to deliver defined numbers of macromolecules into cultured cell lines and primary cells at single molecule resolution. In the nanoinjection platform the nanopipette is used as both a scanning ion conductance microscope (SICM) scanning probe and as an injection probe. The SICM is used to position the nanopipette above the cell surface, before the nanopipette is inserted into the cell into a defined location and to a predefined depth. We demonstrate that the nanoinjection platform enables the quantitative delivery of DNA, globular proteins and protein fibrils into cells with single molecule resolution and that delivery results in a phenotypic change in the cell that depends on the identity of the molecules introduced. Using experiments and computational modelling, we also show that macromolecular crowding in the cell increases the signal to noise ratio for the detection of translocation events, thus the cell itself enhances the detection of the molecules delivered.

biophysics↗

bioRxiv2024

RNA regulates cohesiveness and porosity of a biological condensate

Biological condensates have emerged as key elements of a biological cell function, concentrating disparate biomolecules to accomplish specific biological tasks. RNA was identified as a key ingredient of such condensates, however, its effect on the physical properties of the condensate was found to depend on the condensates composition while its effect on the microstructure has remained elusive. Here, we characterize the physical properties and the microstructure of a protein-RNA condensate by means of large-scale coarse-grained (CG) molecular dynamics simulations. By developing a custom CG model of RNA compatible with a popular CG model of proteins, we systematically investigate the structural, thermodynamic, and kinetic properties of condensate droplets containing thousands of individual protein and RNA molecules over a range of temperatures. While we find RNA to increase the condensates cohesiveness, its effect on the condensates fluidity is more nuanced with longer molecules compacting the condensate and making it less fluid. We show that a biological condensate has a sponge-like morphology of interconnected channels of size that increases with temperature and decreases in the presence of RNA. Our results suggest that longer RNA form a dynamic scaffold within a condensate, regulating not only its fluidity but also permeability to intruder molecules.

biophysics↗

bioRxiv2023

Real-Time Structural Biology of DNA and DNA-Protein Complexes on an Optical Microscope

The intricate interplay between DNA and proteins is key for biological functions such as DNA replication, transcription, and repair. To better understand these interactions, it is crucial to develop tools to study DNA-protein complexes with high spatial and temporal resolution. Here, we use the vertical orientation that DNA adopts on graphene and investigate its interactions with proteins via energy transfer from a probe dye to graphene, achieving spatial resolution down to the [A]ngstrom scale. We measured the bending angle of DNA induced by adenine tracts, bulges, abasic sites and the binding of Escherichia coli endonuclease IV with unprecedented precision and millisecond time resolution. Additionally, we observed the translocation of the O6-alkylguanine DNA alkyltransferase along double-stranded DNA, reaching single-base pair resolution and detecting an affinity for adenine tracts. Overall, we foresee that this method will become a widespread tool for the dynamical study of nucleic acid and nucleic acid-protein interactions.

biophysics↗

bioRxiv2023

Diffusion of DNA on Atomically Flat 2D Material Surfaces

Accurate localization of biomolecules is pivotal for understanding biological processes. Utilizing the atomically flat surface of 2D materials offers a promising route to achieve this without the need for tethering or constraining. Here we comprehensively investigate the binding and diffusion of DNA on hexagonal boron nitride (hBN) surfaces. Our findings reveal non-specific binding of DNA to pristine hBN, with subsequent diffusion and confinement within the 2D plane. Through single-molecule experiments and computational techniques, we explore DNA dynamics, and the effects of defects, step edges and domain boundaries on the motion, which gives insights on the interactions between solid-state surfaces and biomolecules. By engineering a narrow hBN ribbon structure, we enhance confinement, demonstrating its potential in nanofluidic guiding of biomolecules. Our 2D platform serves as a proving ground for next generation high-throughput single-molecule manipulation techniques for enabling applications in biotechnology and nanotechnology.

molecular biology↗

bioRxiv2023

Length-dependent Intramolecular Coil-to-Globule Transition in Poly(ADP-ribose) Induced by Cations

Poly(ADP-ribose) (PAR), a non-canonical nucleic acid, is essential for DNA/RNA metabolism and protein condensation, and its dysregulation is linked to cancer and neurodegeneration. However, key structural insights into PARs functions remain largely uncharacterized, hindered by the challenges in synthesizing and characterizing PAR, which are attributed to its length heterogeneity. A central issue is how PAR, comprised solely of ADP-ribose units, attains specificity in its binding and condensing proteins based on chain length. Here, we integrate molecular dynamics simulations with small-angle X-ray scattering to analyze PAR structures. We reveal the diverse structural ensembles of PAR and the factors influencing them, including a notable length-dependent compaction of PAR upon the addition of small amounts of Mg2+ ions. Unlike PAR15, PAR22 forms ADP-ribose bundles via local intramolecular coil-to-globule transitions. Understanding these length-dependent structural changes could be central to deciphering the specific biological functions of PAR.

biophysics↗

bioRxiv2023

Nanopore translocation reveals electrophoretic force on A- and B-form nucleic acids

Electrophoretic transport plays a pivotal role in advancing sensing technologies. So far, systematic studies have focused on translocation of canonical B-form or A-form nucleic acids, while direct RNA analysis is emerging as the new frontier for nanopore sensing and sequencing. Here, we compare the less-explored dynamics of non-canonical RNA:DNA hybrids in electrophoretic transport with the well-researched transport of B-form DNA. Using DNA/RNA nanotechnology and solid-state nanopores, the translocation of RNA:DNA (RD) and DNA:DNA (DD) duplexes was examined. Notably, RD duplexes were found to translocate through nanopores faster than DD duplexes, despite containing the same number of base pairs. Our experiments reveal that RD duplexes present a non-canonical helix with distinct transport properties from B-form DD molecules. We find RD and DD molecules with the same contour length move with comparable velocity through nanopores. We examined the physical characteristics of both duplex forms using atomic force microscopy, atomistic molecular dynamics simulations, agarose gel electrophoresis, and dynamic light scattering measurements. With the help of coarse-grained and molecular dynamics simulations, we find the effective force per unit length applied by the electric field to a fragment of RD or DD duplex in nanopores with various geometries or shapes to be approximately the same within experimental errors. Our results shed light on the significance of helical form in nucleic acid translocation, with implications for RNA sensing, sequencing, and molecular understanding of electrophoretic transport.

biophysics↗

bioRxiv2023

Benchmarking Molecular Dynamics Force Fields for All-Atom Simulations of Biological Condensates

Proteins containing intrinsically disordered regions are integral components of the cellular signaling pathways and common components of biological condensates. Point mutations in the protein sequence, genetic at birth or acquired through aging, can alter the properties of the condensates, marking the onset of neurodegenerative diseases such as ALS and dementia. While the all-atom molecular dynamics method can, in principle, elucidate the conformational changes that arise from point mutations, the applications of this method to protein condensate systems is conditioned upon the availability of molecular force fields that can accurately describe both structured and disordered regions of such proteins. Using the special-purpose Anton 2 supercomputer, we benchmarked the efficacy of nine presently available molecular force fields in describing the structure and dynamics of a Fused in sarcoma (FUS) protein. Five-microsecond simulations of the full-length FUS protein characterized the effect of the force field on the global conformation of the protein, self-interactions among its side chains, solvent accessible surface area and the diffusion constant. Using the results of dynamic light scattering as a benchmark for the FUS radius of gyration, we identified several force fields that produced FUS conformations within the experimental range. Next, we used these force fields to perform ten-microsecond simulations of two structured RNA binding domains of FUS bound to their respective RNA targets, finding the choice of the force field to affect stability of the RNA-FUS complex. Taken together, our data suggest that a combination of protein and RNA force fields sharing a common four-point water model provides an optimal description of proteins containing both disordered and structured regions and RNA-protein interactions. To make simulations of such systems available beyond the Anton 2 machines, we describe and validate implementation of the best performing force fields in a publicly available molecular dynamics program NAMD. Our NAMD implementation enables simulations of large (tens of millions of atoms) biological condensate systems and makes such simulations accessible to a broader scientific community.

biophysics↗

bioRxiv2022

Long-Range Hopping Conductivity in Proteins

Single molecule measurements show that many proteins, lacking any redox cofactors, nonetheless exhibit electrical conductance on the order of a nanosiemen, implying that electrons can transit an entire protein in less than a nanosecond when subject to a potential difference of less than 1V. In the conventional fast transport scenario where the free energy barrier is zero, the hopping rate is determined by the reorganization energy of approximately 0.8 eV, which sets the time scale of a single hopping event to at least 1s. Furthermore, the Fermi energies of metal electrodes used in experiments are far-removed from the equilibrium redox states of the aromatic residues of the protein, which should additionally slow down the electron transfer. Here, we combine all-atom molecular dynamics (MD) simulations of non-redox active proteins (consensus tetratricopeptide repeats) with an electron transfer theory to demonstrate a molecular mechanism that can account for the unexpectedly fast electron transfer. According to our MD simulations, the reorganization energy produced by the energy shift on charging (the Stokes shift) is close to the conventional value of 0.8 eV. However, the nonergodic sampling of molecular configurations by the protein results in reorganization energies, extracted directly from the distribution of the electrostatic energy fluctuations, that are only ~ 0.2 eV, which is small enough to enable long-range hopping. Using the MD values of the reorganization energies we calculate a current decay with distance that is in agreement with experiment. SignificanceElectron transfer is fundamental to biology, facilitating a range of metabolic processes and efficient energy conversion. Conventionally, electron transfer through proteins is thought to occur via a chain of metal or organic co-factors connecting one side of the protein to another. Recent experiments, however, show that proteins lacking any co-factors can nonetheless transport electrons with high efficiency if properly connected to metal electrodes. This study provides a theoretical model of such cofactor-less transfer, showing that transient occupation of non-equilibrium states of the proteins aromatic residues reduces the barrier to electron hopping, facilitating long range and rapid transport. Our results widen the pool of proteins potentially involved in biological electron transport and provide theoretical underpinning to design of protein molecular electronics.

biophysics↗

bioRxiv2022

DNA double helix, a tiny electromotor

Flowing fluid past curved objects has been used for centuries to power rotary motion in man-made machines. In contrast, rotary motion in nanoscale biological1, 2 or chemical3, 4 systems is produced by biasing Brownian motion through cyclic chemical reactions.5, 6 Here, we show that a curved biological molecule, a DNA or RNA duplex, rotates, unidirectionally, billions of revolutions per minute when electric field is applied along the duplex, with the rotation direction being determined by the duplex chirality. The rotation is found to be powered by the drag force of the electro-osmotic flow, realizing the operating principle of a macroscopic turbine at the nanoscale. The resulting torques are sufficient to power rotation of nanoscale beads and rods, offering an engineering principle for constructing nanoscale systems powered by electric field.

biophysics↗

bioRxiv2022

Lossless end-to-end transport of small molecules through micron-length DNA nanochannels

Designed and engineered protein and DNA nanopores can sense and characterize single molecules and control transmembrane transport of molecular species. However, designed biomolecular pores are less than 100 nm in length and are used primarily for transport across lipid membranes. Nanochannels that span longer distances could be used as conduits for molecules between non-adjacent compartments or cells. Here, we design microns-long, 7 nm diameter DNA nanochannels that small molecules can traverse according to the laws of continuum diffusion. Binding DNA origami caps to channel ends eliminates transport and demonstrates that molecules diffuse from one channel end to the other rather than permeating through channel walls. These micron-length nanochannels can also grow, form interconnects, and interface with living cells. This work thus shows how to construct multifunctional, dynamic agents that control molecular transport, opening new ways of studying intercellular signaling and modulating molecular transport between synthetic and living cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/488239v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@fec963org.highwire.dtl.DTLVardef@157bb83org.highwire.dtl.DTLVardef@d7a274org.highwire.dtl.DTLVardef@ea961c_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

Percolation transition determines protein size limit for passive transport through the nuclear pore complex

Nuclear pore complexes (NPCs) control biomolecular transport in and out of the nucleus. Disordered nucleoporins in the complex"s central pore form a permeation barrier, preventing unassisted transport of large biomolecules. Here, we combine coarse-grained simulations of an experimentally-derived NPC structure with a theoretical model to determine the microscopic mechanism of passive transport. Brute-force simulations of protein diffusion through the NPC reveal telegraph-like behavior, where prolonged diffusion on one side of the NPC is interrupted by rapid crossings to the other. We rationalize this behavior using a theoretical model that reproduces the energetics and kinetics of permeation solely from statistical analysis of transient voids within the disordered mesh. As the protein size increases, the mesh transforms from a soft to a hard barrier, enabling orders-of-magnitude reduction in permeation rate for proteins beyond the percolation size threshold. Our model enables exploration of alternative NPC architectures and sets the stage for uncovering molecular mechanisms of facilitated nuclear transport.

biophysics↗

bioRxiv2021

Single-molecule biophysics experiments in silico: Towards a physical model of a replisome

The interpretation of single-molecule experiments is frequently aided by computational modeling of biomolecular dynamics. The growth of computing power and ongoing validation of computational models suggest that it soon may be possible to replace some experiments out-right with computational mimics. Here we offer a blueprint for performing single-molecule studies in silico using a DNA binding protein as a test bed. We demonstrate how atomistic simulations, typically limited to sub-millisecond durations and zeptoliter volumes, can guide development of a coarse-grained model for use in simulations that mimic experimental assays. We show that, after initially correcting excess attraction between the DNA and protein, qualitative consistency between several experiments and their computational equivalents is achieved, while additionally providing a detailed portrait of the underlying mechanics. Finally the model is used to simulate the trombone loop of a replication fork, a large complex of proteins and DNA.

biophysics↗