Biology subjects

Noy, A.

Publications and source records attributed to Noy, A..

14 recordsLinked to original sources

bioRxiv2026

DNA gyrase in live bacteria forms liquid condensates through weak multivalent bonding of excess GyrB

Type IIA bacterial topoisomerase DNA gyrase, a GyrA/GyrB heterotetramer, has crucial roles maintaining transcription and DNA replication by relaxing positive DNA supercoils through introducing negative supercoils. However, rates of gyrase-catalysed supercoiling in vitro cannot explain much higher rates required in vivo. To address this puzzle, we used high-speed single-molecule fluorescence imaging of GyrA/GyrB reporters in live Escherichia coli, indicating that cells contain [~]40% more GyrB than GyrA expressed in a diffuse pool or in clusters whose mobility depends on whether they are bound to DNA. Unexpectedly, we discovered that clusters are non-stoichiometric containing [~]150% more GyrB than GyrA, significantly greater than the cellular average, with fluorescence recovery after photobleaching revealing that clustered GyrA and GyrB behave as a liquid whose abundance can be increased by applying gyrase-targeting antibiotics. Structural docking indicates that the liquid state is stabilised through excess GyrB progressively binding to existing clusters via weak, multivalent interactions. By operating in liquid condensates, A2B2 that dissociates from DNA can rebind rapidly instead of diffusing away, increasing enzyme processivity to enable multiple rounds of catalysis that can keep pace with transcription and DNA replication in vivo. This demonstrates a new role for condensates of overcoming kinetic limitations imposed by diffusion. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/744598v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1514d4aorg.highwire.dtl.DTLVardef@a95ceeorg.highwire.dtl.DTLVardef@11417daorg.highwire.dtl.DTLVardef@2a2856_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Imaging and mechanism of DNA-DNA recognition mediated by divalent ions

In the cell, DNA must be tightly packed to facilitate its organisation into the nucleus, where recognition of homologous sequences underpins key processes such as recombination. Yet the structural basis of DNA-DNA pairing remains unknown. Here we combine high-resolution AFM and atomistic MD simulations to provide the first direct visualisation of DNA pairing in the presence of divalent ions. We show that strongly paired DNAs often achieve groove-to-groove alignment, driven by ionic bridges connecting the minor grooves of the two duplexes. These contacts are further stabilized by sequence-specific interactions, whose strength and specificity vary with the ion type. This mechanism of ion-mediated groove alignment provides a molecular framework for the long-hypothesized "helical alignment" model, in which homologous recognition is facilitated by preserving structural register between the two helices. Together, our findings reveal a fundamental principle by which divalent ions promote DNA-DNA pairing, with broad implications for chromosomal condensation and genome organization.

biophysics↗

bioRxiv2025

HU searches and binds specific DNA via a multi-step process combining weak electrostatic binding, protein reorientation and DNA flexibility

Despite their limited resolution, coarse-grained simulations have been the chosen method to obtain mechanistic information regarding the process of protein binding to DNA. Here, we demonstrate that state-of-the-art atomic simulations can capture binding events and provide new molecular insight. By using the bacterial model protein HU, we link existing crystal structures to the search and recognition states. We find that weak contacts mediated by only a few positively charged residues enable initial binding, followed by facilitated diffusion and transient rolling on DNA. Extended arms in the HU structure serve as antennae to search for DNA, permitting intrasegmental hops and intersegmental jumps. The transition to specific binding only occurs at the DNA target site, helped by its bendability, indicating a "concerted" binding mechanism between the protein and DNA. We observe a lack of direct binding to the most positively charged area, which is hidden behind HUs arms and defines the recognition state. This prevents the protein from being trapped in random DNA. Instead, the multi-step binding process found here ensures that the high-affinity complex only occurs at the target position. We anticipate that other DNA-binding proteins with multiple surface-charged regions and DNA-bend induction capability might follow a similar strategy.

biophysics↗

bioRxiv2025

Bioelectronic Modulation of Glioblastoma via Wireless Carbon Nanotube Porin Interfaces

The membrane potential (Vmem) and faradaic charge transfer, resulting from altered charge distribution due to ion channels, play a crucial role in cellular bioelectricity. Disruption of Vmem can activate pathways associated with cancer proliferation. Manipulating ion channels may therefore present an effective strategy for treating cancers that fail to respond to conventional therapies. One approach to target these channels, is to manipulate the membrane charge which involves the use of wireless bipolar electrodes such as carbon nanotube porins (CNTPs), which could be inserted into cell membranes to mimic these channels. By utilizing membrane dyes, we observed alterations in Vmem induced by CNTPs and externally applied electric fields. Analyses of cellular behaviors and processes indicated that Vmem is more receptive to stimuli in invasive cancers, while it leads to increased metabolism in less invasive cancers, with notable changes in the cell cycle occurring at approximately 48 hours post-treatment in GBM cell lines. This work shows that CNTPs and electric fields can be used to modulate Vmem and alter cancer cell processes, supporting their potential therapeutic capability.

biophysics↗

bioRxiv2025

Apolipoprotein interaction induces shape remodeling and lipid phase separation in giant unilamellar vesicles

Apolipoprotein A-I (ApoA-I) - a 243-residue amphipathic protein containing an N-terminal globular domain and a primarily helical C-terminal lipid binding domain - is a principal protein component of high-density lipoprotein (HDL) or "good" cholesterol, which is an essential component of lipid homeostasis in humans. Synthesized in the liver and intestine and excreted in the blood, ApoA-I undergoes complex, cooperative, and dynamic self-assembly with membrane lipids, producing unlipi-dated (or weakly lipidated), nascent discoidal, and mature HDL states. In vitro studies demonstrate that the reconstitution of purified protein and lipids restores this cooperative self-assembly. However, the kinetic pathways by which these mesoscopic, proteolipidic assemblies form remain incompletely understood. Here, we monitor the dynamics of ApoA-I-membrane interactions through real-time monitoring of morphological changes, which ensue when ApoA-I is incubated with minimal giant unilamellar vesicles (GUVs) composed of single phospholipids or phase-separating phospholipid-cholesterol mixtures. Our fluorescence microscopy measurements reveal that the interaction initiates a gross, morphological remodeling of the parent vesicle proceeding through discrete stages involving membrane poration, solute leakage, vesiculation, and lipid-lipid phase separation. Our atomic force microscopy measurements confirm that the outcome includes discoidal nanoparticles. This qualitative phenomenology is robust and fully reproducible for different protein mutants and alleles (WT APOA-1, {Delta}49ApoA-I, ApoE-3, and ApoE-4) and other lipid mixtures (including mixtures containing phosphoserine lipids). Our molecular simulations recapitulate the essential shape changes and further reveal the composition dependence of the interactions. Together, these findings outline key steps in protein-lipid interactions that facilitate the assembly of mesoscopic reconstituted lipoproteins and nanodiscs.

biophysics↗

bioRxiv2024

Rep structures can be tuned by ionicity via metastable intermediates in the absence of DNA

DNA helicases undergo conformational changes; however, their structural dynamics are poorly understood. Here, we study single molecules of superfamily 1A DNA helicase Rep, which undergo conformational transitions during bacterial DNA replication, repair and recombination. We use time-correlated single-photon counting (TCSPC), fluorescence correlation spectroscopy (FCS), rapid single-molecule Forster resonance energy transfer (smFRET), Anti-Brownian ELectrokinetic (ABEL) trapping and molecular dynamics simulations (MDS) to provide unparalleled temporal and spatial resolution of Reps domain movements. We detect four states revealing two hitherto hidden intermediates (S2, S3), between the open (S1) and closed (S4) structures, whose stability is salt dependent. Reps open-to-closed switch involves multiple changes to all four subdomains 1A, 1B, 2A and 2B along the S1[->]S2[->]S3[->]S4 transitional pathway comprising an initial truncated swing of 2B which then rolls across the 1B surface, following by combined rotations of 1B, 2A and 2B. High forward and reverse rates for S1[->]S2 suggest that 1B may act to frustrate 2B movement to prevent premature Rep closure in the absence of DNA. These observations support a more general binding model for accessory DNA helicases that utilises conformational plasticity to explore a multiplicity of structures whose landscape can be tuned by salt prior to locking-in upon DNA binding.

biophysics↗

bioRxiv2024

Evaluation in mice of cell-free produced CT584 as a Chlamydia vaccine antigen

Chlamydia trachomatis is the most prevalent bacterial sexually transmitted pathogen worldwide. Since chlamydial infection is largely asymptomatic with the potential for serious complications, a preventative vaccine is likely the most viable long-term answer to this public health threat. Cell-free protein synthesis (CFPS) utilizes the cellular protein manufacturing machinery decoupled from the requirement for maintaining cellular viability, offering the potential for flexible, rapid, and de-centralized production of recombinant protein vaccine antigens. Here, we use CFPS to produce the putative chlamydial type three secretion system (T3SS) needle-tip protein, CT584, for use as a vaccine antigen in mouse models. High-speed atomic force microscopy (HS-AFM) imaging and computer simulations confirm that CFPS-produced CT584 retains a native-like structure prior to immunization. Female mice were primed with CT584 adjuvanted with CpG-1826 intranasally (i.n.) or CpG-1826 + Montanide ISA 720 intramuscularly (i.m.), followed four-weeks later by an i.m. boost before respiratory challenge with 104 inclusion forming units (IFU) of Chlamydia muridarum. Immunization with CT584 generated robust antibody responses but weak cell mediated immunity and failed to protect against i.n. challenge as demonstrated by body weight loss, increased lungs weights and the presence of high numbers of IFUs in the lungs. While CT584 alone may not be the ideal vaccine candidate, the speed and flexibility with which CFPS can be used to produce other potential chlamydial antigens makes it an attractive technique for antigen production.

immunology↗

bioRxiv2024

Correlating fluorescence microscopy, optical and magnetic tweezers to study single chiral biopolymers, tested on DNA plectoneme formation dynamics

Biopolymer topology is critical for determining interactions inside cell environments, exemplified by DNA where its response to mechanical perturbation is as important as biochemical properties to its cellular roles. The dynamic structures of chiral biopolymers exhibit complex dependence with extension and torsion, however the physical mechanisms underpinning the emergence of structural motifs upon physiological twisting and stretching are poorly understood due to technological limitations in correlating force, torque and spatial localization information. We present COMBI-Tweez (Combined Optical and Magnetic BIomolecule TWEEZers), a transformative tool that overcomes these challenges by integrating optical trapping, time-resolved electromagnetic tweezers, and fluorescence microscopy, demonstrated on single DNA molecules, that can controllably form and visualise higher order structural motifs including plectonemes. This technology combined with cutting-edge MD simulations provides quantitative insight into complex dynamic structures relevant to DNA cellular processes and can be adapted to study a range of filamentous biopolymers.

biophysics↗

bioRxiv2023

Atomic description of supercoils and melting bubbles on linear DNA

Although the mechanical response of DNA to physiological torsion and tension is well characterized, the detailed structures are not yet known. By using molecular dynamics simulations on linear DNA with 300 bp, we provide, for the first time, the conformational phase diagram at atomic resolution. Our simulations also reveal the dynamics and diffusion of supercoils. We observe a new state in negative supercoiling, where denaturation bubbles form in AT-rich regions independently of the underlying DNA topology. We thus propose sequence-dependent bubbles could position plectonemes in longer DNA.

biophysics↗

bioRxiv2022

Structural interplay between DNA-shape proteinrecognition and supercoiling: the case of IHF

The integration host factor (IHF) is a prominent example of indirect readout as it imposes one of the strongest bends on relaxed linear DNA. However, the relation between IHF and torsionally constrained DNA, as occurs physiologically, remains unclear. By using atomistic molecular dynamics simulations on DNA minicircles, we reveal, for the first time, the reciprocal influence between a DNA-bending protein and supercoiling. While the increased curvature of supercoiled DNA enhances wrapping around IHF, the protein pins the position of plectonemes, organizing the topology of the loop in a unique and specific manner. In addition, IHF restrains underor overtwisted DNA depending on whether the complex is formed in negatively or positively supercoiled DNA. This effectively enables IHF to become a supercoiling buffer that dampens changes in the surrounding superhelical stress through DNA breathing around the protein or complex dissociation. We finally provide evidence of DNA bridging by IHF and reveal that these bridges divide DNA into independent topological domains. We anticipate that the crosstalk detected here between the active DNA and the multifaceted IHF could be common to other DNA-protein complexes relying on the deformation of DNA.

biophysics↗

bioRxiv2021

Rolling Circle RNA Synthesis Catalysed by RNA

RNA-catalysed RNA replication is widely considered a key step in the emergence of lifes first genetic system. However, RNA replication can be impeded by the extraordinary stability of duplex RNA products, which must be dissociated for re-initiation of the next replication cycle. Here we have explored rolling circle synthesis (RCS) as a potential solution to this strand separation problem. RCS on small circular RNAs - as indicated by molecular dynamics simulations - induces a progressive build-up of conformational strain with destabilisation of nascent strand 5 and 3 ends. At the same time, we observe sustained RCS by a triplet polymerase ribozyme on small circular RNAs over multiple orbits with strand displacement yielding concatemeric RNA products. Furthermore, we show RCS of a circular Hammerhead ribozyme capable of self-cleavage and re-circularisation. Thus, all steps of a viroid-like RNA replication pathway can be catalysed by RNA alone. Our results have implications for the emergence of RNA replication and for understanding the potential of RNA to support complex genetic processes.

biochemistry↗

bioRxiv2020

A nucleoid-associated protein bends and bridges DNA in a multiplicity of topological states with varying specificity

Nucleoid-associated proteins (NAPs) are crucial in organizing prokaryotic DNA and regulating genes. Vital to these activities are complex nucleoprotein structures, however, how these form remains unclear. Integration host factor (IHF) is an Escherichia coli NAP that creates very sharp bends in DNA at sequences relevant to several functions including transcription and recombination, and is also responsible for general DNA compaction when bound non-specifically. We show that IHF-DNA structural multimodality is more elaborate than previously thought, and provide insights into how this drives mechanical switching towards strongly bent DNA. Using single-molecule atomic force microscopy and atomic molecular dynamics simulations we find three binding modes in roughly equal proportions: "associated" (73{degrees} of DNA bend), "half-wrapped" (107{degrees}) and "fully-wrapped" (147{degrees}), only the latter occurring with sequence specificity. We show IHF bridges two DNA double helices through non-specific recognition that gives IHF a stoichiometry greater than one and enables DNA mesh assembly. We observe that IHF-DNA structural multiplicity is driven through non-specific electrostatic interactions that we anticipate to be a general NAP feature for physical organization of chromosomes.

biophysics↗

bioRxiv2020

SerraNA: a program to infer elastic constants from local to global using nucleic acids simulation data

The resistance of DNA to stretch, twist and bend is broadly well estimated by experiments and is important for gene regulation and chromosome packing. However, their sequence-dependence and how bulk elastic constants emerge from local fluctuations is less understood. Here, we present SerraNA, which is an open software that calculates elastic parameters of double-stranded nucleic acids from dinucleotide length up to the whole molecule using ensembles from numerical simulations. The program reveals that global bendability emerge from local periodic bending angles in phase with the DNA helicoidal shape. We also apply SerraNA to the whole set of 136 tetra-bp combinations and we observe a high degree of sequence-dependence for all elastic parameters with differences over 200%. Tetramers with TA and CA base-pair steps are especially flexible, while tetramers containing AA and AT tend to be the most rigid. Our results thus suggest AT-rich motifs generate extreme mechanical properties depending of the exact sequence ordering, which seems critical for creating strong global bendability on longer sequences when phased properly. SerraNA is a tool to be applied in the next generation of interdisciplinary investigations to further understand what determines the elasticity of DNA. Graphical TOC Entry O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/004945v2_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@18aa2ddorg.highwire.dtl.DTLVardef@ca5e02org.highwire.dtl.DTLVardef@1e1febeorg.highwire.dtl.DTLVardef@1b772b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2019

Combining high-resolution AFM with MD simulations shows that DNA supercoiling induces kinks and defects that enhance flexibility and recognition.

In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, we overcome these limitations, by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. We show that the energetics of triplex formation is governed by a delicate balance between electrostatics and bonding interactions. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species.

biophysics↗