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Journal articlePoole W, Ouldridge T, Gopalkrishnan M,
Autonomous learning of generative models with chemical reaction network ensembles
, Journal of the Royal Society Interface, ISSN: 1742-5662Can a micron sized sack of interacting molecules autonomously learn an internalmodel of a complex and fluctuating environment? We draw insights from controltheory, machine learning theory, chemical reaction network theory, and statisticalphysics to develop a general architecture whereby a broad class of chemical systemscan autonomously learn complex distributions. Our construction takes the form ofa chemical implementation of machine learning’s optimization workhorse: gradientdescent on the relative entropy cost function which we demonstrate can be viewedas a form of integral feedback control. We show how this method can be applied tooptimize any detailed balanced chemical reaction network and that the constructionis capable of using hidden units to learn complex distributions.
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Journal articleMukherjee R, Sengar A, Cabello Garcia J, et al., 2024,
Kinetic proofreading can enhance specificity in a non-enzymatic DNA strand displacement network
, Journal of the American Chemical Society, Vol: 146, Pages: 18916-18926, ISSN: 0002-7863Kinetic proofreading is used throughout natural systems to enhance the specificity of molecular recognition. At its most basic level, kinetic proofreading uses a supply of chemical fuel to drive a recognition interaction out of equilibrium, allowing a single free-energy difference between correct and incorrect targets to be exploited two or more times. Despite its importance in biology, there has been little effort to incorporate kinetic proofreading into synthetic systems in which molecular recognition is important, such as nucleic acid nanotechnology. In this article, we introduce a DNA strand displacement-based kinetic proofreading motif, showing that the consumption of a DNA-based fuel can be used to enhance molecular recognition during a templated dimeri zation reaction. We then show that kinetic proofreading can enhance the specificity with which a probe discriminates single nucleo tide mutations, both in terms of the initial rate with which the probe reacts and the long-time behaviour.
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Journal articleJuritz J, Poulton JM, Ouldridge TE, 2022,
Minimal mechanism for cyclic templating of length-controlled copolymers under isothermal conditions
, Journal of Chemical Physics, Vol: 156, ISSN: 0021-9606The production of sequence-specific copolymers using copolymer templates is fundamental to the synthesis of complex biological molecules and is a promising framework for the synthesis of synthetic chemical complexes. Unlike the superficially similar process of self-assembly, however, the development of synthetic systems that implement templated copying of copolymers under constant environmental conditions has been challenging. The main difficulty has been overcoming product inhibition or the tendency of products to adhere strongly to their templates—an effect that gets exponentially stronger with the template length. We develop coarse-grained models of copolymerization on a finite-length template and analyze them through stochastic simulation. We use these models first to demonstrate that product inhibition prevents reliable template copying and then ask how this problem can be overcome to achieve cyclic production of polymer copies of the right length and sequence in an autonomous and chemically driven context. We find that a simple addition to the model is sufficient to generate far longer polymer products that initially form on, and then separate from, the template. In this approach, some of the free energy of polymerization is diverted into disrupting copy–template bonds behind the leading edge of the growing copy copolymer. By additionally weakening the final copy–template bond at the end of the template, the model predicts that reliable copying with a high yield of full-length, sequence-matched products is possible over large ranges of parameter space, opening the way to the engineering of synthetic copying systems that operate autonomously.
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Journal articleMersmann S, Stromich L, Song F, et al., 2021,
ProteinLens: a web-based application for the analysis of allosteric signalling on atomistic graphs of biomolecules
, Nucleic Acids Research, Vol: 49, Pages: W551-W558, ISSN: 0305-1048The investigation of allosteric effects in biomolecular structures is of great current interest in diverse areas, from fundamental biological enquiry to drug discovery. Here we present ProteinLens, a user-friendly and interactive web application for the investigation of allosteric signalling based on atomistic graph-theoretical methods. Starting from the PDB file of a biomolecule (or a biomolecular complex) ProteinLens obtains an atomistic, energy-weighted graph description of the structure of the biomolecule, and subsequently provides a systematic analysis of allosteric signalling and communication across the structure using two computationally efficient methods: Markov Transients and bond-to-bond propensities. ProteinLens scores and ranks every bond and residue according to the speed and magnitude of the propagation of fluctuations emanating from any site of choice (e.g. the active site). The results are presented through statistical quantile scores visualised with interactive plots and adjustable 3D structure viewers, which can also be downloaded. ProteinLens thus allows the investigation of signalling in biomolecular structures of interest to aid the detection of allosteric sites and pathways. ProteinLens is implemented in Python/SQL and freely available to use at: www.proteinlens.io.
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Journal articleSparks H, Dvinskikh L, Firth J, et al., 2020,
Development a flexible light-sheet fluorescence microscope for high-speed 3D imaging of calcium dynamics and 3D imaging of cellular microstructure
, Journal of Biophotonics, Vol: 13, ISSN: 1864-063XWe report a flexible light‐sheet fluorescence microscope (LSFM) designed for studying dynamic events in cardiac tissue at high speed in 3D and the correlation of these events to cell microstructure. The system employs two illumination‐detection modes: the first uses angle‐dithering of a Gaussian light sheet combined with remote refocusing of the detection plane for video‐rate volumetric imaging; the second combines digitally‐scanned light‐sheet illumination with an axially‐swept light‐sheet waist and stage‐scanned acquisition for improved axial resolution compared to the first mode. We present a characterisation of the spatial resolution of the system in both modes. The first illumination‐detection mode achieves dual spectral‐channel imaging at 25 volumes per second with 1024 × 200 × 50 voxel volumes and is demonstrated by time‐lapse imaging of calcium dynamics in a live cardiomyocyte. The second illumination‐detection mode is demonstrated through the acquisition of a higher spatial resolution structural map of the t‐tubule network in a fixed cardiomyocyte cell.
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Journal articleDubois MAJ, Lazaridou A, Choi C, et al., 2019,
Synthesis of 3-Aryl-3-Sulfanyl Azetidines by Iron-Catalyzed Thiol Alkylation with N-Cbz Azetidinols
, Journal of Organic Chemistry, Vol: 84, Pages: 5943-5956, ISSN: 0022-3263New small-ring derivatives can provide valuable motifs in new chemical space for drug design. 3-Aryl-3-sulfanyl azetidines are synthesized directly from azetidine-3-ols in excellent yield by a mild Fe-catalyzed thiol alkylation. A broad range of thiols and azetidinols bearing electron-donating aromatics are successful, proceeding via an azetidine carbocation. The N-carboxybenzyl group is a requirement for good reactivity and enables the NH-azetidine to be revealed. Further reactions of the azetidine sulfides demonstrate their potential for incorporation in drug discovery programs.
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Journal articleSupramaniam P, Ces O, Salehi-Reyhani A, 2019,
Microfluidics for artificial life: techniques for bottom-up synthetic biology
, Micromachines, Vol: 10, Pages: 1-27, ISSN: 2072-666XSynthetic biology is a rapidly growing multidisciplinary branch of science that exploits the advancement of molecular and cellular biology. Conventional modification of pre-existing cells is referred to as the top-down approach. Bottom-up synthetic biology is an emerging complementary branch that seeks to construct artificial cells from natural or synthetic components. One of the aims in bottom-up synthetic biology is to construct or mimic the complex pathways present in living cells. The recent, and rapidly growing, application of microfluidics in the field is driven by the central tenet of the bottom-up approach—the pursuit of controllably generating artificial cells with precisely defined parameters, in terms of molecular and geometrical composition. In this review we survey conventional methods of artificial cell synthesis and their limitations. We proceed to show how microfluidic approaches have been pivotal in overcoming these limitations and ushering in a new generation of complexity that may be imbued in artificial cells and the milieu of applications that result.
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Journal articleGilburt J, Girvan P, Blagg J, et al., 2019,
Ligand discrimination between active and inactive activation loop conformations of Aurora-A kinase is unmodified by phosphorylation
, Chemical Science, Vol: 10, Pages: 4069-4076, ISSN: 2041-6520Structure-based drug design is commonly used to guide the development of potent and specific enzyme inhibitors. Many enzymes – such as protein kinases – adopt multiple conformations, and conformational interconversion is expected to impact on the design of small molecule inhibitors. We measured the dynamic equilibrium between DFG-in-like active and DFG-out-like inactive conformations of the activation loop of unphosphorylated Aurora-A alone, in the presence of the activator TPX2, and in the presence of kinase inhibitors. The unphosphorylated kinase had a shorter residence time of the activation loop in the active conformation and a shift in the position of equilibrium towards the inactive conformation compared with phosphorylated kinase for all conditions measured. Ligand binding was associated with a change in the position of conformational equilibrium which was specific to each ligand and independent of the kinase phosphorylation state. As a consequence of this, the ability of a ligand to discriminate between active and inactive activation loop conformations was also independent of phosphorylation. Importantly, we discovered that the presence of multiple enzyme conformations can lead to a plateau in the overall ligand Kd, despite increasing affinity for the chosen target conformation, and modelled the conformational discrimination necessary for a conformation-promoting ligand.
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Journal articleStorck Saha E, Morales Sanfrutos J, Serwa R, et al., 2019,
Dual chemical probes enable quantitative system-wide analysis of protein prenylation and prenylation dynamics
, Nature Chemistry, Vol: 11, Pages: 552-561, ISSN: 1755-4330Post-translational farnesylation or geranylgeranylation at a C-terminal cysteine residue regulates the localization and function of over 100 proteins, including the Ras isoforms, and is a therapeutic target in diseases including cancer and infection. Here, we report global and selective profiling of prenylated proteins in living cells enabled by the development of isoprenoid analogues YnF and YnGG in combination with quantitative chemical proteomics. Eighty prenylated proteins were identified in a single human cell line, 64 for the first time at endogenous abundance without metabolic perturbation. We further demonstrate that YnF and YnGG enable direct identification of post-translationally processed prenylated peptides, proteome-wide quantitative analysis of prenylation dynamics and alternative prenylation in response to four different prenyltransferase inhibitors, and quantification of defective Rab prenylation in a model of the retinal degenerative disease choroideremia.
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Journal articleHansel C, Crowder S, Cooper S, et al., 2019,
Nanoneedle-mediated stimulation of cell mechanotransduction machinery
, ACS Nano, Vol: 13, Pages: 2913-2019, ISSN: 1936-0851Biomaterial substrates can be engineered to present topographical signals to cells which, through interactions between the material and active components of the cell membrane, regulate key cellular processes and guide cell fate decisions. However, targeting mechanoresponsive elements that reside within the intracellular domain is a concept that has only recently emerged. Here, we show that mesoporous silicon nanoneedle arrays interact simultaneously with the cell membrane, cytoskeleton, and nucleus of primary human cells, generating distinct responses at each of these cellular compartments. Specifically, nanoneedles inhibit focal adhesion maturation at the membrane, reduce tension in the cytoskeleton, and lead to remodeling of the nuclear envelope at sites of impingement. The combined changes in actin cytoskeleton assembly, expression and segregation of the nuclear lamina, and localization of Yes-associated protein (YAP) correlate differently from what is canonically observed upon stimulation at the cell membrane, revealing that biophysical cues directed to the intracellular space can generate heretofore unobserved mechanosensory responses. These findings highlight the ability of nanoneedles to study and direct the phenotype of large cell populations simultaneously, through biophysical interactions with multiple mechanoresponsive components.
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