Biology subjects

Bayley, H.

Publications and source records attributed to Bayley, H..

12 recordsLinked to original sources

bioRxiv2026

Nanopore-based sequence deconvolution of diverse glycosaminoglycans

Glycosaminoglycan (GAG) polysaccharides play vital roles in animal physiology and disease1. Their diverse and intricate patterns of sulfation and epimerization endow them with an extensive potential to encode functional information2,3. GAG characterization, however, remains a formidable challenge for state-of-the-art ensemble-based techniques4,5. Single-molecule techniques are uniquely suited for analysing complex mixtures6,7. Here, we report the single-molecule resolution and counting of diverse GAG di- and oligosaccharides derived from longer heterogeneous chains as part of a deconvolutive nanopore-based workflow that requires no fractionation and is operationally simple. Modular chemical deacylation and amino-selective ring-contractive formation of electrophilic aldehydes enable the parsing of libraries of GAG structures into simplified sets of reactive anhydrosugars for nanopore readout via reversible covalent adduct formation. Discrete clustering of event amplitudes enables direct sugar sizing ([~]10 % step change per residue), which can be coupled to precisely resolved amplitude differences that further reveal sugar fine structure--including the number and position of sulfate groups ([~]2 % step change per sulfate) alongside single- atom stereochemistry ([~]0.5 % step change between epimers). Guided by chemical logic, the reverse mapping of resolved anhydrosugars to their precursors covers [~]84-100 % of all disaccharides and their eliminative digestion variants in natural heparan sulfate (HS). We demonstrate the practical utility and scope of our approach through the compositional analysis of a panel of HS polysaccharides that together encompass natural GAG structural diversity. Moreover, we detect contaminants in heparin, including oversulfated chondroitin sulfate found in an authentic pharmaceutical heparin sample previously implicated in a global healthcare crisis. Together, our results suggest a general chemo-biophysical framework for the precise and sensitive characterization of GAGs that extends to other aminosugar biopolymers. When adapted for portable, widely used nanopore sequencing devices, our approach may offer a path towards the long-sought democratization of glycan analysis.

biochemistry↗

bioRxiv2024

ON-OFF nanopores for optical control of transmembrane ionic communication

[Abstract]Nanoscale photoswitchable proteins could facilitate precise spatiotemporal control of transmembrane communication and support studies in synthetic biology, neuroscience, and bioelectronics. Through covalent modification of the -hemolysin protein pore with arylazopyrazole photoswitches, we have produced "photopores" that transition between iontronic resistor and diode modes in response to irradiation at orthogonal wavelengths. In the diode mode, a low-leak OFF-state nanopore exhibits a reversible increase in unitary conductance of more than 20-fold upon irradiation at 365 nm. A rectification ratio of >5 was achieved with photopores in the diode state by either direct or alternating voltage input. Unlike conventional electronic phototransistors with intensity-dependent photoelectric responses, the photopores regulated current output solely based on the wavelength(s) of monochromatic or dual-wavelength irradiation. Dual-wavelength irradiation at various relative intensities allowed graded adjustment of photopore conductance. By using these properties, photonic signals were converted into ionic signals, highlighting the potential applications of photopores as components of smart devices in synthetic biology.

biophysics↗

bioRxiv2024

Multifunctional Systems in Synthetic Biology: Single-Stranded DNA Signaling for Precise Control of Gene Activation

Controllable gene circuits that respond to defined inputs are essential tools in synthetic biology. By leveraging regulatory mechanisms at either transcriptional or translational levels, synthetic responsive systems have been engineered to recognize diverse signals, such as small molecules (e.g., tetracycline) or physical stimuli (e.g., light). However, these approaches have limitations: small-molecule signals often require high concentrations to be effective, and sophisticated engineering is needed to generate responsive effectors. Here, we establish a simple, versatile gene activation system in which short single-stranded DNAs trigger RNA or protein production by complementing defective single-stranded promoters upstream of target genes. We demonstrate selective gene activation with orthogonal promoters, and logic-gate operations with signal pairs. The signaling system operates in compartmentalized nanoliter droplets scaffolded by bilayers. Signal delivery is controlled by selectively disrupting bilayers or applying transmembrane potential to move signals through protein pores, thereby activating genes within the receiver compartments. This work expands the toolset for engineering multifunctional, responsive materials to meet biotechnological and medical needs, enabling gene activation in response to specific cues.

synthetic biology↗

bioRxiv2024

Targeted, high-resolution sensing of volatile organic compounds by covalent nanopore detection

Volatile organic compounds are choice analytes in a variety of contexts. For example, humans release over 4000 volatile organic compounds, many of which are diagnostic of life-threatening medical conditions. A combination of a large number of potential analytes requires the application of costly, cumbersome technology. Here, we show that covalent nanopore sensing can be used for the targeted detection of a reduced set of analytes in a mixture: in this case aldehydes, which constitute [~]5% of human volatiles. Further, nanopore engineering permits high-resolution detection, which allows closely related aldehydes including isomers to be distinguished. Differential sensing of other chemical classes, such as alcohols, is demonstrated by leveraging their enzymatic conversion to aldehydes. Our approach is compatible with the use of cheap, portable, user-friendly diagnostic devices applicable to a wide variety of objectives, including pollutant monitoring, food and beverage testing and the quality control of pharmaceuticals, as well as disease diagnostics.

biochemistry↗

bioRxiv2024

De novo design of α-helical peptide channels with designer stoichiometry

Despite advances in peptide and protein design, the rational design of membrane-spanning peptides that form conducting channels remains challenging due to our imperfect understanding of the sequence-to-structure relationships that drive membrane insertion, assembly, and conductance. Here, we describe the design and computational and experimental characterization of a series of coiled coil-based peptides that form transmembrane -helical barrels. Through a combination of rational and computational design, we obtain barrels with 5 to 7 helices, as characterized in detergent micelles. In lipid bilayers, these peptide assemblies exhibit two conductance states with relative populations dependent on the applied potential: (i) a low-conductance states that correlate with variations in the modeled coiled-coil barrel geometries, indicating stable transmembrane -helical barrels; and (ii) high-conductance states in which single pores change size in discrete steps. Notably, the high-conductance states are similar for all peptides in contrast to the low-conductance states. This indicates the formation of large, dynamic pores through the recruitment and expulsion of peptides, as observed in natural barrel-stave peptide pores. These findings establish rational routes to design and tune functional membrane-spanning peptide channels with specific conductance and geometry.

synthetic biology↗

bioRxiv2024

Location of phosphorylation sites within long polypeptide chains by binder-assisted nanopore detection

The detection and mapping of protein phosphorylation sites are essential for understanding the mechanisms of various cellular processes and for identifying targets for drug development. The study of biopolymers at the single-molecule level has been revolutionized by nanopore technology. In this study, we detect protein phosphorylation within long polypeptides (>600 amino acids), after the attachment of binders that interact with phosphate monoesters; electro-osmosis is used to drive the tagged chains through engineered protein nanopores. By monitoring the ionic current carried by a nanopore, phosphorylation sites are located within individual polypeptide chains, providing a valuable step toward nanopore proteomics.

biochemistry↗

bioRxiv2023

A 3D microtumour system that faithfully represents ovarian cancer minimal residual disease

BackgroundBulk cancer and minimal residual disease (MRD) are characterised by different molecular drivers and therefore necessitate different therapeutic strategies. However, there are currently no 3D models that can faithfully recapitulate MRD ex vivo for therapy development. MethodsA microfluidic technique was implemented to construct 3D microtumours, in which tumour cells, either by themselves or with fibroblasts, were encapsulated in viscous hydrogels. The 3D microtumours were analysed for their response to first-line chemotherapeutics and characterised through RNA-Seq, by comparing them to both 2D cultures and clinical samples. ResultsOur microfluidic platform guarantees the fabrication of 3D microtumours of tailorable size and cell content, which recreate key features of tumours such as hypoxia, characteristic organization of the cytoskeleton and a dose-response to chemotherapeutics close to the physiological range. The 3D microtumours were also used to examine non-genetic heterogeneity in ovarian cancer and could fully reflect the recently described "Oxford Classic" five molecular signatures. The gene expression profile of 3D microtumours following chemotherapy treatment closely resembled that of MRD in ovarian cancer patients, showing the upregulation of genes involved in fatty acid metabolism. We demonstrate that these 3D microtumours are ideal for drug development by showing how they support the identification of a promising inhibitor of fatty acid oxidation, perhexiline, which specifically targets chemotherapy-resistant MRD ovarian cancer cells and not bulk cancer cells. ConclusionWe have obtained the first 3D model of ovarian cancer MRD by using microtumours generated through microfluidics. This system is ideal for high-throughput drug screening and, given its versatility, it can be readily extended to additional types of cancer, as well as accommodate multiple cell types to generate complex tumour microenvironments.

cancer biology↗

bioRxiv2023

Enzyme-less nanopore detection of post-translational modifications within long polypeptides

Means to sequence DNA and RNA quickly and cheaply have revolutionized biology and medicine. The ability to analyse cellular proteins and their millions of variants would be an advance of comparable importance, but requires a fresh technical approach. We use electroosmosis for the non-enzymatic capture, unfolding and translocation of individual polypeptides of more than 1200 residues by a protein nanopore. By monitoring the ionic current carried by the nanopore, we locate post-translational modifications deep within the polypeptide chains, and thereby lay the groundwork for obtaining inventories of the proteoforms in cells and tissues.

biochemistry↗

bioRxiv2022

Functional Integration of 3D-Printed Cerebral Cortical Tissue into a Brain Lesion

Engineering human tissue with diverse cell types and desired cellular architectures and functions is a considerable challenge. The cerebral cortex, which has a layered cellular architecture composed of layer-specific neurons organised into vertical columns, delivers higher cognition through intricately wired neural circuits. However, current tissue engineering approaches cannot produce such structures. Here, we use a droplet printing technique to fabricate tissues comprising simplified cerebral cortical columns. Human induced pluripotent stem cells (hiPSCs) were differentiated into upper- and deep-layer neural progenitors, which were then printed to form cerebral cortical tissues with a two-layer organization. The tissues showed layer-specific biomarker expression and developed an integrated network of processes. Implantation of the printed cortical tissues into mouse brain explants resulted in substantial implant-host integration across the tissue boundaries as demonstrated by the projection of processes, the migration of neurons and the appearance of correlated Ca2+ signals. The approach we have developed might be used for the evaluation of drugs and nutrients that promote tissue integration. Importantly, our approach might be applied in personalised implantation treatments that restore the cellular structure and function of a damaged brain by using 3D tissues derived from a patients own iPSCs.

bioengineering↗

bioRxiv2021

A lipid-based parallel processor for chemical signals

A key goal of bottom-up synthetic biology is to construct cell- and tissue-like structures. Underpinning cellular life is the ability to process several external chemical signals, often in parallel. Until now, however, cell- and tissue-like structures have only been constructed with one signalling pathway. Here, we construct a dual-signal processor from the bottom up in a modular fashion. The processor comprises three aqueous compartments bounded by lipid bilayers and operates in an aqueous environment. It can receive two chemical signals from the external environment, process them orthogonally, and then produce a distinct output for each signal. It is suitable for both sensing and enzymatic processing of environmental signals with fluorescence and molecular outputs. In the future, such processors could serve as smart drug delivery vehicles or as modules within synthetic tissues to control their behaviour in response to external chemical signals.

synthetic biology↗

bioRxiv2020

Droplet printing reveals the importance of micron-scale structure for bacterial ecology

Bacteria often live in diverse communities where the spatial arrangement of strains and species is considered critical for their ecology, including whether strains can coexist, which are ecologically dominant, and how productive they are as a community1,2. However, a test of the importance of spatial structure requires manipulation at the fine scales at which this structure naturally occurs3-8. Here we develop a droplet-based printing method to arrange different bacterial genotypes across a sub-millimetre array. We use this to test the importance of fine-scale spatial structure by printing strains of the gut bacterium Escherichia coli that naturally compete with one another using protein toxins9,10. This reveals that the spatial arrangement of bacterial genotypes is important for ecological outcomes. Toxin-producing strains largely eliminate susceptible non-producers when genotypes are well-mixed. However, printing strains side-by-side creates an ecological refuge such that susceptible strains can coexist with toxin producers, even to the extent that a susceptible strain outnumbers the toxin producer. Head-to-head competitions between toxin producers also reveals strong effects, where spatial structure can make the difference between one strain winning and mutual destruction. Finally, we print different potential barriers between two competing strains to understand why space is so important. This reveals the importance of processes that limit the free diffusion of molecules. Specifically, we show that cells closest to a toxin producer bind to and capture toxin molecules, which creates a refuge for their clonemates. Our work provides a new method to generate customised bacterial communities with defined spatial distributions, and reveals that micron-scale changes in these distributions can drive major shifts in their ecology.

microbiology↗

bioRxiv2019

Direct detection of molecular intermediates from first-passage times

All natural phenomena are governed by energy landscapes. However, the direct measurement of this fundamental quantity remains challenging, particularly in complex systems involving intermediate states. Here, we uncover key details of the energy landscapes that underpin a range of experimental systems through quantitative analysis of first-passage time distributions. By combined study of colloidal dynamics in confinement, transport through a biological pore and the folding kinetics of DNA hairpins, we demonstrate conclusively how a short-time, power-law regime of the first-passage time distribution universally reflects the number of intermediate states associated with each process, irrespective of the lengthscales, timescales or interactions in the system. We thereby establish a powerful method for investigating the underlying mechanisms of complex molecular processes.

biophysics↗