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Journal articleJurinović K, Mitra M, Mukherjee R, et al., 2026,
Design of DNA strand displacement reactions
, Current Opinion in Biotechnology, Vol: 97, ISSN: 0958-1669DNA strand displacement (SD) reactions are central to the operation of many synthetic nucleic acid systems, including molecular circuits, sensors, and machines. Over the years, a broad set of design frameworks has emerged to accommodate various functional goals, initial configurations, and environmental conditions. Nevertheless, key challenges persist, particularly in reliably predicting reaction kinetics. In contrast to reviews centred on network-level architectures, this article focuses on the design and analysis of individual SD reactions, highlighting kinetic mechanisms, structural determinants, and the current limits of predictive modelling. We identify promising innovations while analysing the factors that continue to hinder predictive accuracy. We conclude by outlining future directions for achieving more robust and programmable behaviour in DNA-based systems.
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Journal articleCollins K, Stanley CE, Ouldridge TE, 2025,
Biochemical surface patterning in microfluidic devices
, Current Opinion in Biotechnology, Vol: 96, ISSN: 0958-1669The capacity to pattern biomolecules within microfluidic devices expands the scope of microfluidic technologies. In such patterned systems, surface-bound components remain localized, while the microfluidic network supplies reagents and removes waste products. This approach has enabled continuous protein expression from patterned DNA, chemical synthesis from immobilized enzymes, and cell capture assays. Here, we review methods to pattern surfaces within microfluidic devices. Patterns may be printed before or after the device is assembled; pre-bonding methods are compatible with well-established open-surface patterning protocols but present challenges for device bonding and alignment. Conversely, post-bonding methods are compatible with standard bonding procedures but rely on less established, sequential patterning protocols. Future progress will require consistent reporting of pattern signal and noise relative to controls.
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Journal articleQureshi B, Poulton J, Ouldridge T, 2025,
Thermodynamic limits on general far-from-equilibrium molecular templating networks
, Newton, ISSN: 2950-6360Cells maintain a highly specific, far-from-equilibrium population of RNA and protein molecules. They do so via complex reaction networks in which templates catalyse the assembly of desired products. We show that information transmission from templates to products in such networks is bounded by functions of the maximal difference in free-energy changes between assembly path-ways. Surprisingly, putative systems operating at the bounds do not have a high net flux around the network, as is typical in far-from-equilibrium systems and observed in biology. Instead, the upper bound on accuracy for a given network structure is achieved in “pseudo-equilibrium”. Here, each product is produced and degraded by time-reversed trajectories along a single (product-specific) pathway with negligible entropy production; product yields are determined by the free-energy changes along those pathways. The limit imposed by these free-energy changes induces a thermodynamic constraint on accuracy, even if a single templating process is arbitrarily kinetically selective.
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Journal articleGuntoro J, Ouldridge T, 2025,
The role of sequence information in minimal models of molecular assembly
, Journal of Physics: Complexity, Vol: 6, ISSN: 2632-072XSequence-directed assembly processes – such as protein folding – allow the assembly of a large number of structures with high accuracy from only a small handful of fundamental building blocks. We aim to explore how efficiently sequence information can be used to direct assembly by studying variants of the temperature-1 abstract tile assembly model (aTAM). We ask whether, for each variant, there exists a finite set of tile types that can deterministically assemble any shape producible by agiven assembly model; we call such tile type sets “universal assembly kits”. Our first model, which we call the “backboned aTAM”, generates backbone-assisted assembly by forcing tiles to be added to lattice positions neighbouring the immediately preceding tile, using a predetermined sequence of tile types. We demonstrate the existence of universal assembly kit for the backboned aTAM, and show that the existence of this set is maintained even under stringent restrictions to the rules of assembly. We compare these results to a less constrained model that we call sequenced aTAM, which also uses a predetermined sequence of tiles, but does not constrain a tile to neighbour the immediately preceding tiles. We prove that this model has no universal assembly kit in the stringent case. The lack of such a kit is surprising, given that the number of tile sequences of length N scales faster than both the number and worst-case Kolmogorov complexity of producible shapes of size N for a sufficiently large – butfinite – set of tiles. Our results demonstrate the importance of physical mechanisms, and specifically geometric constraints, in facilitating efficient use of the information in molecular programs for structure assembly.
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Journal articleCabello-Garcia J, Mukherjee R, Bae W, et al., 2025,
Information propagation through enzyme-free catalytic templating of DNA dimerization with weak product inhibition
, Nature Chemistry, ISSN: 1755-4330Information propagation by sequence-specific, template-catalyzed molecular assembly is a key motif facilitating life’s biochemical complexity, allowing the production ofthousands of sequence-defined proteins from only 20 distinct building blocks. By contrast, exploitation of catalytic templating is rare in non-biological contexts, particularlyin enzyme-free environments, where even the template-catalyzed formation of dimers is challenging. The main obstacle is product inhibition: the tendency of products to bind to templates more strongly than individual monomers, preventing catalytic turnover. We present a rationally designed enzyme-free system in which a DNA template catalyzes, with weak product inhibition, the production of sequence-specific DNA dimers. We demonstrate selective templating of 9 different dimers with high specificity and catalytic turnover; we then show that the products can participate in downstream reactions, and that the dimerization can be coupled to covalent bond formation. Most importantly, our mechanism demonstrates a rational design principle for engineering information propagation by molecular templating.
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Journal articleGuntoro JEB, Qureshi BJ, Ouldridge TE, 2025,
The interplay of heterogeneity and product detachment in templated polymer copying
, Journal of Chemical Physics, Vol: 162, ISSN: 0021-9606Templated copolymerization, in which information stored in the sequence of a heteropolymer template is copied into another polymer product, is the mechanism behind all known methods of genetic information transfer. A key aspect of templated copolymerization is the eventual detachment of the product from the template. A second key feature of natural biochemical systems is that the template-binding free energies of both correctly matched and incorrect monomers are heterogeneous. Previous work has considered the thermodynamic consequences of detachment and the consequences of heterogeneity for polymerization speed and accuracy, but the interplay of both separation and heterogeneity remains unexplored. In this work, we investigate a minimal model of templated copying that simultaneously incorporates both detachment from behind the leading edge of the growing copy and heterogeneous interactions. We first extend existing coarse-graining methods for models of polymerization to allow for heterogeneous interactions. We then show that heterogeneous copying systems with explicit detachment do not exhibit the subdiffusive behavior observed in the absence of detachment when near equilibrium. Next, we show that heterogeneity in correct monomer interactions tends to result in slower, less accurate copying, while heterogeneity in incorrect monomer interactions tends to result in faster, more accurate copying, due to an increased roughness in the free energy landscape of either correct or incorrect monomer pairs. Finally, we show that heterogeneity can improve on known thermodynamic efficiencies of homogeneous copying, but these increased thermodynamic efficiencies do not always translate to increased efficiencies of information transfer.
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Journal articlePoole W, Ouldridge T, Gopalkrishnan M, 2025,
Autonomous learning of generative models with chemical reaction network ensembles
, Journal of the Royal Society Interface, Vol: 22, 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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