Periodic Reporting for period 3 - RESSPICAC (Rational Engineering of Synthetic Systems for Propagation of Information via Catalytic Assembly of Copies)
Okres sprawozdawczy: 2022-11-01 do 2024-04-30
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 will explore minimal theoretical models of copying, leveraging recent advances in the thermodynamics of small, fluctuating systems and the thermodynamics of information processing, to identify the aforementioned underlying principles. We will then translate this insight, via detailed molecular simulation and experimental characterisation of novel reaction motifs, into the construction of minimal synthetic copying systems.
The project will provide insight into analogous copying processes in living organisms, and shed light on primitive living systems. It will also lay the groundwork for engineering synthetic systems with this key cell-like ability, a step on the road to building synthetic living systems. Additionally, it will be a first step towards using synthetic copying outside of living sytems to produce novel chemicals. Theory and simulation will drive the experiments, making rational design of systems possible whilst providing insight into the fundamental thermodynamics of information processing and computation, and the biophysics of novel nucleic acid interactions. Indeed, designing and building concrete molecular systems based on fundamental theory will enhance our understanding of the theories themselves.
In this project, my team will explore minimal theoretical models of copying, leveraging recent advances in the thermodynamics of small, fluctuating systems and the thermodynamics of information processing, to identify the aforementioned underlying principles. We will then translate this insight, via detailed molecular simulation and experimental characterisation of novel reaction motifs, into the construction of minimal synthetic copying systems.
The project will provide insight into analogous copying processes in living organisms, and shed light on primitive living systems. It will also lay the groundwork for engineering synthetic systems with this key cell-like ability, a step on the road to building synthetic living systems. Additionally, it will be a first step towards using synthetic copying outside of living sytems to produce novel chemicals. Theory and simulation will drive the experiments, making rational design of systems possible whilst providing insight into the fundamental thermodynamics of information processing and computation, and the biophysics of novel nucleic acid interactions. Indeed, designing and building concrete molecular systems based on fundamental theory will enhance our understanding of the theories themselves.
The majority of our work so far has revolved around developing a novel DNA-based reaction motif that allows for copying of sequence information in DNA templates, without the use of highly-evolved enzymes. Fundamental theoretical work from our 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 the template as it forms. We have successfully demonstrated an experimental realisation of this motif, using the tools of synthetic DNA nanotechnology [J. Cabello Garcia et al., ACS Nano 2021]. We have demonstrated the ability to produce copies of dimer templates with high accuracy [J. Cabello Garcia, PhD thesis 2022, also presented at the 3rd Workshop on Stochastic Thermodynamics (2022) by T. E. Ouldridge and with a manuscript in preparation]. We have also shown that this motif, which we call handhold mediated strand displacement (HMSD) can be used inconjunction with the biologically prevalent scheme of kinetic proofreading [presented at FDN2022 By R. Mukherjee, manuscript in preparation]. Kinetic proofreading describes the use of molecular fuel to enhance the accuracy of a biological process; it is extremely common in nature, but we believe that our work is the first demonstration of the motif in a synthetic context.
Alongside this experimental work, we have been conducting theory and simulation to guide the next steps of the project. We have simulated the HMSD motif, identifying important differences in the underlying process from similar motifs in DNA nanotechnology [manuscript in preparation]. As part of this process, we produced an extensive review on how the coarse-grained model oxDNA can be used as a simulation tool [A. Sengar et al., Front. Mol. Biosci. 2021]. We have also developed a general theoretical framework that allows us to analyse models of complex molecular copying processes in a much more efficient way than was previously possible [Presented at the 3rd Workshop on Stochastic Thermodynamics (2022) by B. Qureshi, manuscript in preparation].
Alongside this experimental work, we have been conducting theory and simulation to guide the next steps of the project. We have simulated the HMSD motif, identifying important differences in the underlying process from similar motifs in DNA nanotechnology [manuscript in preparation]. As part of this process, we produced an extensive review on how the coarse-grained model oxDNA can be used as a simulation tool [A. Sengar et al., Front. Mol. Biosci. 2021]. We have also developed a general theoretical framework that allows us to analyse models of complex molecular copying processes in a much more efficient way than was previously possible [Presented at the 3rd Workshop on Stochastic Thermodynamics (2022) by B. Qureshi, manuscript in preparation].
The HMSD motif represents a truly novel approach to implementing molecular copying. Fundamentally, it has the potential to overcome the main challenge of molecular copying, which is a phenomenon known as "product inhibition". The units within a template need to bind strongly to the units that assemble into the copy in order for the copying to happen. However, this strong binding becomes a problem once the copy is formed; in a naive approach, the newly-formed copy binds even more strongly to the template than the original units from which it was formed. This problem only gets worse as the polymer to be copied gets longer, and it has prevented the engineering of synthetic copying systems. During this project we have demonstrated, both in principle and in practice, how this problem of product inhibition might be overcome in a simple process. This is a major step beyond the start of the art in the field.
From now until the end of the project, we expect the following results.
- Completion and publication of the sub-projects noted above, especially: demonstration of dimer copying using HMSD; the first demonstration of kinetic proofreading in a synthetic context; the development of a framework for efficient analysis of complex models of polymer copying; and the detailed molecular simulation of the HMSD process.
- Application of the intial results of the project to demonstrate copying of templates of length 3, 4 and possibly longer.
- Further theoretical/simulation insight into the key features of successful copying processes, in particular the use of HMSD in more complex settings and the adaptation of our theoretical framework to short molecules.
- A modification of the HMSD mechanism to allow it to act as the core of a self-replicating system. Self-replication is a type of copying in which the product can itself act as a template for the production of more templates.
From now until the end of the project, we expect the following results.
- Completion and publication of the sub-projects noted above, especially: demonstration of dimer copying using HMSD; the first demonstration of kinetic proofreading in a synthetic context; the development of a framework for efficient analysis of complex models of polymer copying; and the detailed molecular simulation of the HMSD process.
- Application of the intial results of the project to demonstrate copying of templates of length 3, 4 and possibly longer.
- Further theoretical/simulation insight into the key features of successful copying processes, in particular the use of HMSD in more complex settings and the adaptation of our theoretical framework to short molecules.
- A modification of the HMSD mechanism to allow it to act as the core of a self-replicating system. Self-replication is a type of copying in which the product can itself act as a template for the production of more templates.