Topics

Recent Highlights

17/07/2020 In situ generation of RNA complexes for synthetic molecular strand displacement circuits in autonomous systems submitted

Synthetic molecular circuits implementing DNA or RNA strand-displacement reactions can be used to build complex systems such as molecular computers and feedback control systems. Despite recent advances, application of nucleic acid-based circuits in vivo remains challenging due to a lack of efficient methods to produce their essential components - multi-stranded complexes known as 'gates' − in situ, i.e. in living cells or other autonomous systems. Here, we propose the use of naturally occurring self-cleaving ribozymes to cut a single-stranded RNA transcript into a gate complex of shorter strands, thereby opening new possibilities for the autonomous and continuous production of RNA strands in a stoichiometrically and structurally controlled way.

02/07/2020 Implementing Non-Equilibrium Networks with Active Circuits of Duplex Catalysts accepted at DNA26

DNA strand displacement (DSD) reactions have been used to construct chemical reaction networks in which species act catalytically at the level of the overall stoichiometry of reactions. These effective catalytic reactions are typically realised through one or more of the following: many-stranded gate complexes to coordinate the catalysis, indirect interaction between the catalyst and its substrate, and the recovery of a distinct ``catalyst'' strand from the one that triggered the reaction. These facts make emulation of the out-of-equilibrium catalytic circuitry of living cells more difficult. Here, we propose a new framework for constructing catalytic DSD networks: Active Circuits of Duplex Catalysts (ACDC). ACDC components are all double-stranded complexes, with reactions occurring through 4-way strand exchange. Catalysts directly bind to their substrates, and and the ``identity'' strand of the catalyst recovered at the end of a reaction is the same molecule as the one that initiated it. We analyse the capability of the framework to implement catalytic circuits analogous to phosphorylation networks in living cells. We also propose two methods of systematically introducing mismatches within DNA strands to avoid leak reactions and introduce driving through net base pair formation. We then combine these results into a compiler to automate the process of designing DNA strands that realise any catalytic network allowed by our framework.

09/06/2020 Modelling DNA-strand displacement reactions in the presence of base-pair mismatches accepted by J. Am. Chem. Soc.

Toehold-mediated strand displacement is the most abundantly used method to achieve dynamic switching in DNA-based nanotechnology. An ‘invader’ strand binds to the ‘toehold’ overhang of a target strand and replaces a target-bound ’incumbent’ strand. Hereby, complementarity of the invader to the single-stranded toehold provides the energetic bias of the reaction. Despite the widespread use of strand displacement reactions for realizing dynamic DNA nanostructures, variants on the basic motif have not been completely characterized. Here we introduce a simple thermodynamic model, which is capable of quantitatively describing the kinetics of strand displacement reactions in the presence of mismatches, using a minimal set of parameters. Furthermore, our model highlights that base pair fraying and internal loop formation are important mechanisms when involving mismatches in the displacement process. Our model should provide a helpful tool for the rational design of strand-displacement reaction networks.

02/06/2020 Handhold-mediated strand displacement: a nucleic acid-based mechanism for generating far-from-equilibrium assemblies through templated reactions submitted.

Toehold-mediated strand displacement (TMSD) is a nucleic acid-based reaction wherein an invader strand (I) replaces an incumbent strand (N) in a duplex with a target strand (T). TMSD is driven by toeholds, overhanging single-stranded domains in T recognised by I. Although TMSD is responsible for the outstanding potential of dynamic DNA nanotechnology, TMSD cannot implement templating, the central mechanism by which biological systems generate complex, far-from equilibrium assemblies like RNA or proteins. Therefore, we introduce handhold-mediated strand displacement (HMSD). Handholds are toehold analogues located in N and capable of implementing templating. We measure the kinetics of 98 different HMSD systems to demonstrate that handholds can accelerate the rate of invader-target (IT) binding by more than 4 orders of magnitude. Furthermore, handholds of moderate length accelerate reactions whilst allowing detachment of the product IT from N. We are thus able to experimentally demonstrate the use of HMSD-based templating to produce highly-specific far-from-equilibrium DNA duplexes. Check out Javi's talk at FNANO, or a simplified discussion of the overall project

02/06/2020 Edge-effects dominate copying thermodynamics for finite-length molecular oligomers submitted.

Living systems produce copies of information-carrying molecules such as DNA by assembling monomer units into finite-length oligomer (short polymer) copies. We explore the role of initiation and termination of the copy process in the thermodynamics of copying. By splitting the free-energy change of copy formation into informational and chemical terms, we show that copy accuracy plays no direct role in the overall thermodynamics. Instead, it is thermodynamically costly to produce outputs that are more similar to the oligomers in the environment than sequences obtained by randomly sampling monomers. Copy accuracy can be thermodynamically neutral, or even favoured, depending on the surroundings. Oligomer copying mechanisms can thus function as information engines that interconvert chemical and information-based free energy. Hard thermodynamic constraints on accuracy derived for infinite-length polymers instead manifest as kinetic barriers experienced while the copy is template-attached. These barriers are easily surmounted by shorter oligomers.

02/06/2020 Design of hidden thermodynamic driving for non-equilibrium systems via mismatch elimination during DNA strand displacement published in Nat. Comms. 

Recent years have seen great advances in the development of synthetic self-assembling molecular systems. Designing out-of-equilibrium architectures, however, requires a more subtle control over the thermodynamics and kinetics of reactions. We propose a mechanism for enhancing the thermodynamic drive of DNA strand-displacement reactions whilst barely perturbing forward reaction rates: the introduction of mismatches within the initial duplex. Through a combination of experiment and simulation, we demonstrate that displacement rates are strongly sensitive to mismatch location and can be tuned by rational design. By placing mismatches away from duplex ends, the thermodynamic drive for a strand-displacement reaction can be varied without significantly affecting the forward reaction rate. This hidden thermodynamic driving motif is ideal for the engineering of non-equilibrium systems that rely on catalytic control and must be robust to leak reactions.

 

 

Functional molecular systems

Biochemical networks within cells achieve remarkable functionality, including sensing, signalling, information-processing and replication. We aim to understand the fundamental physical principles that set the scope of this behaviour, allowing development of engineering principles for artificial analogs of these systems.

Since these systems of interest typically involve small numbers of molecules, randomness plays an important role. This work often touches on the deep connections between two bodies of work that deal explicitly with randomness: information theory, which describes the role of randomness in communication, and statistical mechanics, through which randomness is related to thermodynamics.

Recently, we've started to translate our basic understanding into the engineering of synthetic, nucleic acid-based analogs of these natural systems within our own lab here at Imperial. To get a taste, check out Javi's talk at FNANO2020, or a simplified discussion of his overall project

Relevant Publications

  1. Bae W, Stan GBV, Ouldridge TE, "In situ generation of RNA complexes for synthetic molecular strand displacement circuits in autonomous systems", submitted. 
  2. Cabello-Garcia J, Bae W, Stan GBV, Ouldridge TE, "Handhold-mediated strand displacement: a nucleic acid-based mechanism for generating far-from-equilibrium assemblies through templated reactions" submitted.
  3. Poulton JM, Ouldridge TE, "Edge-effects dominate copying thermodynamics for finite-length molecular oligomers"submitted.
  4. Lankinen A, Mullor Ruiz I, Ouldridge TE, "Implementing Non-Equilibrium Networks with Active Circuits of Duplex Catalysts", submitted. 
  5. Plesa T, Stan G-BV, Ouldridge TE, Bae W, "Robust control of biochemical reaction networks via stochastic morphing" submitted.
  6. Ouldridge TE, Brittain RA, ten Wolde PR, 2018, The power of being explicit: demystifying work, heat, and free energy in the physics of computation, in "The Energetics of Computing in Life and Machines", SFI Press.
  7. Deshpande A, Ouldridge TE, 2019, "Optimizing enzymatic catalysts for rapid turnover of substrates with low enzyme sequestration" submitted.
  8. Brittain RA, Jones NS, Ouldridge TE, 2019, Biochemical Szilard engines for memory-limited inference, New J. Phys. 21,063022.
  9. Poulton J, ten Wolde PR and Ouldridge TE, 2019, Non-equilibrium correlations in minimal dynamical models of polymer copying, PNAS: 116,1946-1951.
  10. Deshpande A, Ouldridge  TE, 2017, High rates of fuel consumption are not required by insulating motifs to suppress retroactivity in biochemical circuitsEngineering Biology: 1,86-99.
  11. Poole W, Ortiz-Muñoz A, Behera A, Jones NS,  Ouldridge  TE, Winfree E, Gopalkrishnan M, 2017, Chemical Boltzmann MachinesIn DNA Computing and Molecular Programming. DNA 2017. Lecture Notes in Computer Science: 10467,210-231.
  12. Brittain RA, Jones NS, Ouldridge TE, 2017, What we learn from the learning rateJ. Stat. Mech.: 063592.
  13. Ouldridge TE, 2017, The importance of thermodynamics for molecular systems, and the importance of molecular systems for thermodynamicsNat. Comput.: in press.
  14. Ouldridge TE, ten Wolde PR, 2017, Fundamental costs in the production and destruction of persistent polymer copies, Phys. Rev. Lett.: 118,158103.
  15. Ouldridge TE, Govern CC, ten Wolde PR, 2017, Thermodynamics of computational copying in biochemical systemPhys. Rev. X: 7,021004.
  16. McGrath T, Jones NS, ten Wolde PR, Ouldridge TE, 2017, Biochemical machines for the interconversion of mutual information and workPhys. Rev. Lett.: 118,028101.
  17. ten Wolde PR, Becker NB, Ouldridge TE, Mugler A, 2015, Fundamental Limits to Cellular SensingJ. Stat. Phys.: 162,1395-1424.
  18. Ouldridge TE, ten Wolde PR, 2014, The robustness of proofreading to crowding-induced pseudo-processivity in the MAPK pathwayBiophysical Journal: 107,2425-2435.

Coarse-grained modelling of DNA

The elegant selectivity of Watson-Crick base-pairing makes DNA an extremely useful tool for the construction of nanoscale objects and machines. Stable structures and mechanical cycles can be programmed into a system of single strands by careful choice of the sequences of bases. I'm particularly interested in using nucleic acids to design artificial analogs of complex cellular systems, to enable careful exploration of the design principles and engineering possibilities.

Despite the experimental successes, there is no clear theoretical description of the processes involved. We have developed a nucleotide-level coarse grained model of DNA, oxDNA, which is detailed enough to capture the essential physics of assembly processes, yet simple enough to be applicable over long time scales. Code, user guides and examples for simulating the model can be downloaded from this site.

The oxDNA model was developed in the Doye / Louis groups in Oxford. It has since been applied in collaboration with the Turberfield group in Oxford, the Winfree group in Caltech and the Nir group at the Ben-Gurion University of Negev, as well as being used independently by other researchers.

Even with oxDNA, it is still not practical to simulate the formation of very large structures. A collaboration with the Turberfield and Kwiatkowska groups in Oxford has led to a less detailed model that can describe the formation of DNA origami structures.

Relevant Publications

  1. Irmisch P, Ouldridge TE, Seidel R, 2020, "Modelling DNA-strand displacement reactions in the presence of base-pair mismatches"in press at J. AM. Chem. Soc. 
  2. Haley NEC, Ouldridge TE, Mullor Ruiz I, Geraldini A, Louis AA, Bath J and Turberfield AA, 2020, Design of hidden thermodynamic driving for non-equilibrium systems via mismatch elimination during DNA strand displacement, Nat. Comms. 11:2562.
  3. Lankinen A, Mullor Ruiz I, Ouldridge TE, "Implementing Non-Equilibrium Networks with Active Circuits of Duplex Catalysts", submitted. 
  4. Fonseca P, Romano F, Schreck JS, Ouldridge TE, Doye JPK and Louis AA, 2018, Multi-scale coarse-graining for the study of assembly pathways in DNA-brick self assembly, J. Chem. Phys. 148:134910. 
  5. Khara DC, Schreck JS, Tomov TE, Berger Y, Ouldridge TE, Doye JPK and Nir E, 2018, DNA bipedal motor walking dynamics: an experimental and theoretical study of the dependency on step size, Nucl. Acids Res. 46:1553-1561.
  6. Snodin BEK, Romano F, Rovigatti L, Ouldridge TE, Louis AA, and Doye JPK, 2016, Direct Simulation of the Self-Assembly of a Small DNA Origami, ACS Nano: 10,1724-1737.
  7. Dunn KE, Dannenberg F, Ouldridge TE, Kwiatkowska M, Turberfield AJ, Bath J, 2015, Guiding the folding pathway of DNA origami, Nature: 525,82-86.
  8. Dannenberg F, Dunn KE, Bath J, Kwiatkowska M, Turberfield AJ, Ouldridge TE, 2015, Modelling DNA Origami Self-Assembly at the Domain Level, J. Chem. Phys.: 143,165102.
  9. Snodin BEK, Randisi F, Mosayebi M, Sulc P, Schreck JS, Romano F, Ouldridge TE, Tsukanov R, Nir E, Louis AA, Doye JPK, 2015, Introducing improved structural properties and salt dependence into a coarse-grained model of DNA, J. Chem. Phys.: 142,234901.
  10. Schreck JS, Ouldridge TE, Romano F, Sulc P, Shaw L, Louis AA, Doye JPK, 2015, DNA hairpins primarily promote duplex melting rather than inhibiting hybridization, Nucleic Acids Research: 43,6181-6190.
  11. Mosayebi M, Louis AA, Doye JPK, Ouldridge TE, 2015, Force-induced rupture of a DNA duplex, ACS Nano: 9,11993-12003.
  12. Machinek RR, Ouldridge TE, Haley NE, Bath J, Turberfield AJ, 2014, Programmable energy landscapes for kinetic control of DNA strand displacement, Nature Communications: 5,5324.
  13. Doye JPK, Ouldridge TE, Louis AA, Romano F, Sulc P, Matek C, Snodin BEK, Rovigatti L, Schreck JS, Harrison RM, Smith WPJ, 2013, Coarse-graining DNA for simulations of DNA nanotechnology, Physical Chemistry Chemical Physics: 15,20395-20414.
  14. Srinivas N, Ouldridge TE, Sulc P, Schaeffer JM, Yurke B, Louis AA, Doye JPK, Winfree E, 2013, On the biophysics and kinetics of toehold-mediated DNA strand displacement, Nucleic Acids Research: 41,10641-10658.
  15. Ouldridge TE, Sulc P, Romano F, Doye JPK, Louis AA, 2013, DNA hybridization kinetics: Zippering, internal displacement and sequence dependence, Nucleic Acids Research: 41,8886-8895.
  16. Ouldridge TE, Hoare RL, Louis AA, Doye JPK, Bath J, Turberfield AJ, 2013, Optimizing DNA nanotechnology through coarse-grained modeling: A two-footed DNA walker, ACS Nano: 7,2479-2490.
  17. Sulc P, Romano F, Ouldridge TE, Rovigatti L, Doye JPK, Louis AA, 2012, Sequence-dependent thermodynamics of a coarse-grained DNA model, Journal of Chemical Physics: 137,135101.
  18. Ouldridge TE, Louis AA, Doye JPK, 2011, Structural, mechanical, and thermodynamic properties of a coarse-grained DNA model, Journal of Chemical Physics: 134,085101.
  19. Ouldridge TE, Louis AA, Doye JPK, 2010, DNA nanotweezers studied with a coarse-grained model of DNA, Physical Review Letters: 104,178101.

Thermodynamics of small systems

Thermodynamics, the science of heat and energy transfer, emerged as a field in the 19th century, motivated by the need to describe the engines that powered the industrial revolution. One of the challenges of modern science is to adapt and extend the theory to describe microscopic systems in which fluctuations play a key role. Biological and biologically-inspired systems are a key arena for these new ideas, due both to the need to understand natural molecular analogues of the engines and processes that we are familiar with at much larger length scales, and the possibility of developing artificial devices ourselves.

Not only does thermodynamics provide understanding of biological systems, but the study of real biophysical devices in turn provides us with a deeper understanding of the thermodynamic principles at play. In particular, the natural diffusive behaviour of biomolecules allows us to study complex systems that do not require external manipulation to function.

Relevant Publications

  1. Poulton JM, Ouldridge TE, "Edge-effects dominate copying thermodynamics for finite-length molecular oligomers"submitted.
  2. Ouldridge TE, 2020, A biochemical device To demystify a century-old thermodynamics puzzle from theoretical physics, Reseach Outreach: 112. 
  3. Brittain RA, Jones NS, Ouldridge TE, 2019, Biochemical Szilard engines for memory-limited inference, in press at New. J. Phys.
  4. Ouldridge TW, Brittain RA, ten Wolde PR, 2019, The power of being explicit: demystifying work, heat, and free energy in the physics of computation, in "The Energetics of Computing in Life and Machines", SFI Press.
  5. Stopnitzky E, Still S, Ouldridge TE and Altenberg L, 2019,  Physical Limitations of Work Extraction from Temporal Correlations, Phys. Rev. E: 99,042115. 
  6. Poulton J, ten Wolde PR and Ouldridge TE, 2019, Non-equilibrium correlations in minimal dynamical models of polymer copying, PNAS: 116,1946-1951.
  7. Deshpande A, Ouldridge  TE, 2017, High rates of fuel consumption are not required by insulating motifs to suppress retroactivity in biochemical circuitsEngineering Biology: 1,86-99.
  8. Deshpande A, Gopalkrishnan M, Ouldridge TE, Jones NS, 2017,  Designing the Optimal Bit: Balancing Energetic Cost, Speed and ReliabilityProc. Roy. Soc. A.: 473,20170117.
  9. Brittain RA, Jones NS, Ouldridge TE, 2017, What we learn from the learning rateJ. Stat. Mech.: 063592.
  10. Ouldridge TE, 2017, The importance of thermodynamics for molecular systems, and the importance of molecular systems for thermodynamicsNat. Comput.: in press.
  11. Ouldridge TE, ten Wolde PR, 2017, Fundamental costs in the production and destruction of persistent polymer copiesPhys. Rev. Lett.: 118,158103.
  12. Ouldridge TE, Govern CC, ten Wolde PR, 2017, Thermodynamics of computational copying in biochemical systemPhys. Rev. X: 7,021004.
  13. McGrath T, Jones NS, ten Wolde PR, Ouldridge TE, 2017, Biochemical machines for the interconversion of mutual information and workPhys. Rev. Lett.: 118,028101.

Simulation tools and algorithms

Our work often involves systems that are too complex to be treated analytically. This means that simulations are a key tool in our research, and we are interested in simulation techniques and analysis tools for systems involving biomolecular reactions.

In this work we collaborate with the Doye / Louis groups in Oxford, the biochemical networks group of Pieter Rein ten Wolde in Amsterdam, Michael Tretyakov in Nottingham and Ruslan Davidchack in Leicester, and Oliver Henrich in Strathclyde.

Relevant Publications

  1. Henrich O, Yair AGF, Curk T, Ouldridge TE, 2018, Coarse-grained simulation of DNA using LAMMPS, Eur. Phys. J. E., 41: 57.
  2. Davidchack RL, Ouldridge TE, Tretyakov MV, 2017, Geometric integrator for Langevin systems with quaternion-based rotational degrees of freedom and hydrodynamic interactionsJ. Chem. Phys., 147:224103.
  3. Vijaykumar A, Ouldridge TE, ten Wolde PR, Bolhuis PG 2017, Multiscale simulations of anisotropic particles combining Brownian Dynamics and Green's Function Reaction DynamicsJournal of Chemical Physics: 146,114106.
  4. Davidchack RL, Ouldridge TE, Tretyakov MV, 2015, New Langevin and Gradient Thermostats for Rigid Body DynamicsJournal of Chemical Physics: 142,144114.
  5. Ouldridge TE, 2012, Inferring bulk self-assembly properties from simulations of small systems with multiple constituent species and small systems in the grand canonical ensembleJournal of Chemical Physics: 137,144105.
  6. Ouldridge TE, Louis AA, Doye JPK, 2010, Extracting bulk properties of self-assembling systems from small simulationsJournal of Physics: Condensed Matter: 22,104102.

Biomolecular Engineering

We've recently acquired our own (small) lab within the synthetic biology space at Imperial. We're using this lab to actually put our theoretical ideas into practice, engineering functional molecular systems from nucleic acids. These systems are both useful test-beds for our theory and engineering platforms for synthetic biology.

Our work currently focusses on engineering non-equilibrium information processing systems, analogs of the signalling and transcription/translation machinery in cells.  To get a taste, check out Javi's talk at FNANO2020, or a simplified discussion of his overall project

Relevant Publications

  1. Bae W, Stan GBV, Ouldridge TE, "In situ generation of RNA complexes for synthetic molecular strand displacement circuits in autonomous systems", submitted. 
  2. Cabello-Garcia J, Bae W, Stan GBV, Ouldridge TE, "Handhold-mediated strand displacement: a nucleic acid-based mechanism for generating far-from-equilibrium assemblies through templated reactions" submitted.