Periodic Reporting for period 4 - RESSPICAC (Rational Engineering of Synthetic Systems for Propagation of Information via Catalytic Assembly of Copies)
Periodo di rendicontazione: 2024-05-01 al 2025-12-31
Biological information is stored in the sequence of units found in long polymers like DNA, RNA and proteins. Copying this information from one polymer to another is vital to life: the "central dogma" of molecular biology describes how a sequence of nucleotides in DNA is copied into a sequence of nucleotides in a newly-assembled RNA polymer, and then into a sequence of amino acids in a protein. These processes involve sophisticated machinery in living organisms, but even the most highly evolved is bound by the same physical principles as simpler systems that must have operated in early life. Unfortunately, we do not understand these basic principles that enable accurate and reliable copies to be made of polymer templates, as evidenced by the fact that we cannot build minimal synthetic copying systems.
In this project my team aimed to provide insight into the fundamental principles of copying processes in living systems, and use that insight to engineer synthetic, enzyme-free copying systems in the laboratory. We aimed to lay the groundwork for engineering for building synthetic living systems that exploit this mechanism, and to provide a first step towards using synthetic copying outside of living systems to produce novel chemicals.
My team worked at three levels. Most fundamentally, we explored minimal theoretical models of copying, leveraging recent advances in the thermodynamics of small, fluctuating systems and the thermodynamics of information processing. We developed new approaches for analysing theoretical models of copying, and used those methods to explore the basic principles of copying systems, uncovering potential mechanisms for effective copying and deriving relationships between the the accuracy and the necessary costs of copying. At an intermediate level, we modelled specific processes based on the interactions of synthetic DNA strands that allowed us to translate these fundamental insights into practical designs of enzyme-free, synthetic copying systems. Finally, we tested these designs in wet-lab experiments, successfully demonstrating information propagation by templated copying and realising the kinetic proofreading motif (an important part of natural copying systems) in a synthetic system for the first time.
In conclusion, the project succeeded in advancing our understanding of the fundamental principles of templated copying, and in implementing those principles in the design of a synthetic copying system.
In this project my team aimed to provide insight into the fundamental principles of copying processes in living systems, and use that insight to engineer synthetic, enzyme-free copying systems in the laboratory. We aimed to lay the groundwork for engineering for building synthetic living systems that exploit this mechanism, and to provide a first step towards using synthetic copying outside of living systems to produce novel chemicals.
My team worked at three levels. Most fundamentally, we explored minimal theoretical models of copying, leveraging recent advances in the thermodynamics of small, fluctuating systems and the thermodynamics of information processing. We developed new approaches for analysing theoretical models of copying, and used those methods to explore the basic principles of copying systems, uncovering potential mechanisms for effective copying and deriving relationships between the the accuracy and the necessary costs of copying. At an intermediate level, we modelled specific processes based on the interactions of synthetic DNA strands that allowed us to translate these fundamental insights into practical designs of enzyme-free, synthetic copying systems. Finally, we tested these designs in wet-lab experiments, successfully demonstrating information propagation by templated copying and realising the kinetic proofreading motif (an important part of natural copying systems) in a synthetic system for the first time.
In conclusion, the project succeeded in advancing our understanding of the fundamental principles of templated copying, and in implementing those principles in the design of a synthetic copying system.
We have developed and applied enzyme-free, DNA-based reaction motifs that allow for copying of sequence information in DNA templates. Theoretical work from my group [J. Juritz et al., J. Chem. Phys. 2022] showed that a deceptively simple mechanism allows, in principle, for the reliable production of polymer copies from molecular templates. At the heart of that mechanism is a motif in which the copy polymer rips itself off from the template as it forms. We demonstrated that such a motif can be engineered using synthetic DNA alone [J. Cabello Garcia et al., ACS Nano 2021], and then that two variants can be used to produce copies of dimer templates with high accuracy [J. Cabello Garcia et al., Nature Chemistry 2025, Mitra et al., submitted to J. Am. Chem. Soc]. By the end of the project, we had successfully shown that this basic idea can be applied to the copying of trimeric, tetrameric and pentameric templates [Mukherjee et al., manuscript in preparation - results presented at Foundations of Nanoscience 2025 and Functional DNA Nanotechnology 2025]. We have also shown that this mechanism can be used in conjunction with the biologically prevalent scheme of kinetic proofreading [Mukherjee et al., J. Am. Chem. Soc. 2024], wherein a molecular fuel is used to enhance the specificity of a biological process. Although kinetic proofreading is extremely common in nature, our work is the first demonstration of the celebrated motif in a synthetic context.
Alongside this experimental work, we pursued theory and simulation to guide the experiments and explore the principles of molecular templating more generally. We have simulated key DNA reaction mechanisms, identifying important differences in the underlying process relative to similar approaches in DNA nanotechnology [Bakhtawar et al, Nano Lett. 2026, Stannard et al. and Sengar et al., latter two manuscripts in preparation and presented at oxDNA users and developers workshop 2026]. We have developed two frameworks that allow for improved theoretical analysis of models of copying processes in different contexts [Qureshi et al., J. Chem. Phys. 2023 and Guntoro et al., J. Chem. Phys. 2025], and applied them to exemplar models. We have also resolved a long-standing paradox by explaining how accurate information propagation in arbitrarily complex molecular templating networks is constrained by the thermodynamic properties of those networks [Qureshi et al., Newton 2026].
As part of this process, and to help disseminate our results, we produced extensive reviews on how the coarse-grained model oxDNA can be used as a simulation tool [A. Sengar et al., Front. Mol. Biosci. 2021] and on the design of systems based on DNA strand displacement [Jurinovic et al., Curr. Op. Biotech. 2026]. As a result of our paper on kinetic proofreading, we were invited to write a "News and Views" for Nature Nanotechnology on enhancing recognition beyond equilibrium in synthetic systems [Ouldridge et al., Nat. Nano., 2025]. In addition, my team has given talks at 36 different scientific events, and poster presentations at a number of others, reaching an estimate 4500 people. We have also sought to reach out beyond academia, communicating directly with an estimated 500 members of the general public through outreach activities and also recording a number of publicly available videos.
The work performed here has led to additional funding from the EIC, the Royal Society and the Defence Science and Technology Laboratory to explore variant molecular templating systems. We have also submitted a patent based on an auto-catalytic extension to the templating mechanism.
Alongside this experimental work, we pursued theory and simulation to guide the experiments and explore the principles of molecular templating more generally. We have simulated key DNA reaction mechanisms, identifying important differences in the underlying process relative to similar approaches in DNA nanotechnology [Bakhtawar et al, Nano Lett. 2026, Stannard et al. and Sengar et al., latter two manuscripts in preparation and presented at oxDNA users and developers workshop 2026]. We have developed two frameworks that allow for improved theoretical analysis of models of copying processes in different contexts [Qureshi et al., J. Chem. Phys. 2023 and Guntoro et al., J. Chem. Phys. 2025], and applied them to exemplar models. We have also resolved a long-standing paradox by explaining how accurate information propagation in arbitrarily complex molecular templating networks is constrained by the thermodynamic properties of those networks [Qureshi et al., Newton 2026].
As part of this process, and to help disseminate our results, we produced extensive reviews on how the coarse-grained model oxDNA can be used as a simulation tool [A. Sengar et al., Front. Mol. Biosci. 2021] and on the design of systems based on DNA strand displacement [Jurinovic et al., Curr. Op. Biotech. 2026]. As a result of our paper on kinetic proofreading, we were invited to write a "News and Views" for Nature Nanotechnology on enhancing recognition beyond equilibrium in synthetic systems [Ouldridge et al., Nat. Nano., 2025]. In addition, my team has given talks at 36 different scientific events, and poster presentations at a number of others, reaching an estimate 4500 people. We have also sought to reach out beyond academia, communicating directly with an estimated 500 members of the general public through outreach activities and also recording a number of publicly available videos.
The work performed here has led to additional funding from the EIC, the Royal Society and the Defence Science and Technology Laboratory to explore variant molecular templating systems. We have also submitted a patent based on an auto-catalytic extension to the templating mechanism.
We have proposed the first minimal mechanisms by which effective catalytic templating can occur, exploring the regimes in which such mechanisms are functional. Our ideas -- which have subsequently been validated by experiment -- for overcoming the main challenge of templated copying, a phenomenon known as "product inhibition", represent a significant advance.
Our theoretical work has also resolved a long-standing paradox surrounding molecular templating networks. It has long been suspected that the transfer of information from catalytic templates to products must be associated with a minimal thermodynamic cost. We showed that the accuracy with which a specific set of products can be maintained by an arbitrary templating network is, indeed, constrained by its thermodynamic properties. Surprisingly, the operation of the most efficient templating networks is completely different from the behaviour observed in nature: products are disassembled via a reversal of the pathway by which they were formed. The observation that this novel pathway is optimal is a major advance in our understanding.
To assist with these theoretical investigations, we have developed a number of tools for the analysis of minimal models of (templated) copolymerisation processes. These tools allow certain models to be efficiently analysed, without resorting to simulation, which was previously impossible. They therefore represent a significant advance in theoretical capabilities.
The Handhold-Mediated Strand Displacement (HMSD) motif, which was developed as part of this project and underlies our synthetic molecular templating experiments, is a novel mechanism that allows new functionality in DNA nanotechnology. Its unusual topology, wherein a strand A recognises a strand B but then pulls a complementary strand C off from B, is unlike anything previously introduced in DNA nanotechnology. We have introduced and thoroughly characterised this mechanism, allowing its use by the community.
In this project we used the HMSD motif to demonstrate information propagation during catalytic molecular templating. We showed, for the first time, how product inhibition could be suppressed during dimerisation, enabling the templates to function effectively as catalysts. This achievement allowed us to extend the mechanism to sequence-specific templating of trimer, tetramer and pentamer formation -- a feat that has never previously been demonstrated.
We also demonstrated that HMSD-based processes can be integrated with a kinetic proofreading mechanism, allowing chemical free energy to be spent to improve the accuracy of molecular recognition. Kinetic proofreading is a common motif in natural settings, but our work was the first time it has been demonstrated in a synthetic system. It therefore represents a substantial advance in the state of the art.
Our theoretical work has also resolved a long-standing paradox surrounding molecular templating networks. It has long been suspected that the transfer of information from catalytic templates to products must be associated with a minimal thermodynamic cost. We showed that the accuracy with which a specific set of products can be maintained by an arbitrary templating network is, indeed, constrained by its thermodynamic properties. Surprisingly, the operation of the most efficient templating networks is completely different from the behaviour observed in nature: products are disassembled via a reversal of the pathway by which they were formed. The observation that this novel pathway is optimal is a major advance in our understanding.
To assist with these theoretical investigations, we have developed a number of tools for the analysis of minimal models of (templated) copolymerisation processes. These tools allow certain models to be efficiently analysed, without resorting to simulation, which was previously impossible. They therefore represent a significant advance in theoretical capabilities.
The Handhold-Mediated Strand Displacement (HMSD) motif, which was developed as part of this project and underlies our synthetic molecular templating experiments, is a novel mechanism that allows new functionality in DNA nanotechnology. Its unusual topology, wherein a strand A recognises a strand B but then pulls a complementary strand C off from B, is unlike anything previously introduced in DNA nanotechnology. We have introduced and thoroughly characterised this mechanism, allowing its use by the community.
In this project we used the HMSD motif to demonstrate information propagation during catalytic molecular templating. We showed, for the first time, how product inhibition could be suppressed during dimerisation, enabling the templates to function effectively as catalysts. This achievement allowed us to extend the mechanism to sequence-specific templating of trimer, tetramer and pentamer formation -- a feat that has never previously been demonstrated.
We also demonstrated that HMSD-based processes can be integrated with a kinetic proofreading mechanism, allowing chemical free energy to be spent to improve the accuracy of molecular recognition. Kinetic proofreading is a common motif in natural settings, but our work was the first time it has been demonstrated in a synthetic system. It therefore represents a substantial advance in the state of the art.