Design of hidden thermodynamic driving for non-equilibrium systems via mismatch elimination during DNA strand displacement

Nature Communications Springer Nature 11 (2020) 2562

NEC Haley, TE Ouldridge, I Mullor Ruiz, A Geraldini, A Louis, J Bath, AJ Turberfield

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.

Design, optimization and analysis of large DNA and RNA nanostructures through interactive visualization, editing and molecular simulation

Nucleic Acids Research Oxford University Press (OUP) (2020)

E Poppleton, J Bohlin, M Matthies, S Sharma, F Zhang, P Šulc

<jats:title>Abstract</jats:title> <jats:p>This work seeks to remedy two deficiencies in the current nucleic acid nanotechnology software environment: the lack of both a fast and user-friendly visualization tool and a standard for structural analyses of simulated systems. We introduce here oxView, a web browser-based visualizer that can load structures with over 1 million nucleotides, create videos from simulation trajectories, and allow users to perform basic edits to DNA and RNA designs. We additionally introduce open-source software tools for extracting common structural parameters to characterize large DNA/RNA nanostructures simulated using the coarse-grained modeling tool, oxDNA, which has grown in popularity in recent years and is frequently used to prototype new nucleic acid nanostructural designs, model biophysics of DNA/RNA processes, and rationalize experimental results. The newly introduced software tools facilitate the computational characterization of DNA/RNA designs by providing multiple analysis scripts, including mean structures and structure flexibility characterization, hydrogen bond fraying, and interduplex angles. The output of these tools can be loaded into oxView, allowing users to interact with the simulated structure in a 3D graphical environment and modify the structures to achieve the required properties. We demonstrate these newly developed tools by applying them to design and analysis of a range of DNA/RNA nanostructures.</jats:p>

Controlling the bioreceptor spatial distribution at the nanoscale for single molecule counting in microwell arrays

ACS Sensors American Chemical Society 4 (2019) 2327-2335

D Daems, I Rutten, J Bath, D Decrop, H Van Gorp, E Pérez Ruiz, S De Feyter, A Turberfield, J Lammertyn

The ability to detect low concentrations of protein biomarkers is crucial for the early-stage detection of many diseases and therefore indispensable for improving diagnostic devices for healthcare. Here, we demonstrate that by integrating DNA nanotechnologies like DNA origami and aptamers, we can design innovative biosensing concepts for reproducible and sensitive detection of specific targets. DNA origami structures decorated with aptamers were studied as a novel tool to structure the biosensor surface with nanoscale precision in a digital detection bioassay, enabling control of the density, orientation, and accessibility of the bioreceptor to optimize the interaction between target and aptamer. DNA origami was used to control the spatial distribution of an in-house-generated aptamer on superparamagnetic microparticles, resulting in an origami-linked digital aptamer bioassay to detect the main peanut antigen Ara h1 with 2-fold improved signal-to-noise ratio and 15-fold improved limit of detection compared to a digital bioassay without DNA origami. Moreover, the sensitivity achieved was 4 orders of magnitude higher than commercially available and literature-reported enzyme-linked immunosorbent assay techniques. In conclusion, this novel and innovative approach to engineer biosensing interfaces will be of major interest to scientists and clinicians looking for new molecular insights and ultrasensitive detection of a broad range of targets, and, for the next generation of diagnostics.

Modifying Membrane Morphology and Interactions with DNA Origami Clathrin-Mimic Networks.

ACS nano (2019)

CMA Journot, V Ramakrishna, MI Wallace, AJ Turberfield

We describe the triggered assembly of a bio-inspired DNA origami meshwork on a lipid membrane. DNA triskelia, three-armed DNA origami nanostructures inspired by the membrane-modifying protein clathrin, are bound to lipid mono- and bi-layers using cholesterol anchors. Polymerization of triskelia, triggered by the addition of DNA staples, links triskelion arms to form a mesh. Using transmission electron microscopy, we observe nanoscale local deformation of a lipid monolayer induced by triskelion polymerization that is reminiscent of the formation of clathrin-coated pits. We also show that the polymerization of triskelia bound to lipid bilayers modifies interactions between them, inhibiting the formation of a synapse between giant unilamellar vesicles and a supported lipid bilayer.

Peptide Assembly Directed and Quantified Using Megadalton DNA Nanostructures.

ACS nano (2019)

J Jin, EG Baker, CW Wood, J Bath, DN Woolfson, AJ Turberfield

In nature, co-assembly of polypeptides, nucleic acids and polysaccharides is used to create functional supramolecular structures. Here we show that DNA nanostructures can be used to template interactions between peptides, and to enable the quantification of multivalent interactions that would otherwise not be observable. Our functional building blocks are DNA-peptide hybrids comprising de novo designed dimeric coiled-coil peptides covalently linked to oligonucleotide tags. These hybrids are incorporated in megadalton DNA origami nanostructures and direct nanostructure association through peptide-peptide interactions. Free and bound nanostructures can be counted directly from electron micrographs allowing estimation of the dissociation constants of the peptides linking them. Results for a single peptide-peptide interaction are consistent with measured solution-phase free energies; DNA nanostructures displaying multiple peptides allow the effects of polyvalency to be probed. This use of DNA nanostructures as identifiers allows the binding strengths of homo- and hetero-dimeric peptide combinations to be measured in a single experiment and gives access to dissociation constants that are too low to be quantified by conventional techniques. The work also demonstrates that hybrid biomolecules can be programmed to achieve spatial organization of complex synthetic biomolecular assemblies.

Targeting 3D Bladder Cancer Spheroids with Urease-Powered Nanomotors

ACS Nano American Chemical Society (ACS) 13 (2019) 429-439

AC Hortelão, R Carrascosa, N Murillo-Cremaes, T Patiño, S Sánchez

Dimensions and Global Twist of Single-Layer DNA Origami Measured by Small-Angle X-Ray Scattering.

ACS nano (2018)

MAB Baker, AJ Tuckwell, JF Berengut, J Bath, F Benn, AP Duff, AE Whitten, KE Dunn, RM Hynson, AJ Turberfield, LK Lee

The rational design of complementary DNA sequences can be used to create nanostructures that self-assemble with nanometer precision. DNA nanostructures have been imaged by atomic force microscopy and electron microscopy. Small-angle X-ray scattering (SAXS) provides complementary structural information on the ensemble-averaged state of DNA nanostructures in solution. Here we demonstrate that SAXS can distinguish between different single-layer DNA origami tiles that look identical when immobilized on a mica surface and imaged with atomic force microscopy. We use SAXS to quantify the magnitude of global twist of DNA origami tiles with different crossover periodicities: these measurements highlight the extreme structural sensitivity of single-layer origami to the location of strand crossovers. We also use SAXS to quantify the distance between pairs of gold nanoparticles tethered to specific locations on a DNA origami tile and use this method to measure the overall dimensions and geometry of the DNA nanostructure in solution. Finally, we use indirect Fourier methods, which have long been used for the interpretation of SAXS data from biomolecules, to measure the distance between DNA helix pairs in a DNA origami nanotube. Together, these results provide important methodological advances in the use of SAXS to analyze DNA nanostructures in solution and insights into the structures of single-layer DNA origami.

Chiral DNA origami nanotubes with well‐defined and addressable inside and outside surfaces

Angewandte Chemie International Edition Wiley‐VCH Verlag 57 (2018) 7687-7690

F Benn, NEC Haley, AE Lucas, E Silvester, S Helmi, R Schreiber, J Bath, AJ Turberfield

We report the design and assembly of chiral DNA nanotubes with well‐defined and addressable inside and outside surfaces. We demonstrate that the outside surface can be functionalised with a chiral arrangement of gold nanoparticles to create a plasmonic device and that the inside surface can be functionalised with a track for a molecular motor allowing transport of a cargo within the central cavity.

Self-propulsion of catalytic nanomotors synthesised by seeded growth of asymmetric platinum–gold nanoparticles

Chemical Communications Royal Society of Chemistry 54 (2018) 1901-1904

I Santiago, L Jiang, J Foord, A Turberfield

Asymmetric bimetallic nanomotors are synthesised by seeded growth in solution, providing a convenient and high-throughput alternative to the usual top-down lithographic fabrication of self-propelled catalytic nanoparticles. These synthetic nanomotors catalyse H2O2 decomposition and exhibit enhanced diffusion that depends on fuel concentration, consistent with their chemical propulsion.

Lipid Bilayer Modulation using DNA Origami Mimics of Clathrin

BIOPHYSICAL JOURNAL 114 (2018) 103A-103A

V Ramakrishna, C Journot, AJ Turberfield, MI Wallace

DNA origami nanostructured surfaces for enhanced detection of molecular interactions

22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018 1 (2018) 16-19

D Daems, I Rutten, W Pfeifer, D Decrop, D Spasic, J Bath, B Saccà, A Turberfield, J Lammertyn

Copyright© (2018) by Chemical and Biological Microsystems Society.All rights reserved. The performance of biosensors strongly depends on the nanoarchitecture of the biosensing surface. In many studies the bioreceptor density, orientation and accessibility are often overlooked, resulting in suboptimal biosensing devices. Here, DNA origami structures were decorated with aptamers and studied as a novel tool to structure the biosensor surface with nanoscale precision, favoring interaction between target and aptamer. Using this novel method to engineer biosensing interfaces of two in-house developed biosensing platforms, we were able to accurately detect the presence of a specific target and to compete with existing biosensors in reproducibility, SNR and LOD, without the need for backfilling.

Practical aspects of structural and dynamic DNA nanotechnology

MRS Bulletin 42 (2017) 889-896

P Wang, G Chatterjee, H Yan, TH Labean, AJ Turberfield, CE Castro, G Seelig, Y Ke

© Copyright Materials Research Society 2017. DNA nanostructures are a set of materials with well-defined physical, chemical, and biological properties that can be used on their own or incorporated with other materials for many applications. Herein, the practical aspects of utilizing DNA nanostructures (structural or dynamic) as materials are comprehensively covered. This article first summarizes properties of DNA molecules and practical considerations and then discusses the fundamental design principles of structural DNA nanostructures. Finally, various aspects of dynamic DNA nanostructure-based actuation and computation are included.

The evolution of DNA-templated synthesis as a tool for materials discovery

Accounts of Chemical Research American Chemical Societ 50 (2017) 2496-2509

RK O'Reilly, A Turberfield, TR Wilks

Precise control over reactivity and molecular structure is a fundamental goal of the chemical sciences. Billions of years of evolution by natural selection have resulted in chemical systems capable of information storage, self-replication, catalysis, capture and production of light, and even cognition. In all these cases, control over molecular structure is required to achieve a particular function: without structural control, function may be impaired, unpredictable, or impossible. The search for molecules with a desired function is often achieved by synthesizing a combinatorial library, which contains many or all possible combinations of a set of chemical building blocks (BBs), and then screening this library to identify "successful" structures. The largest libraries made by conventional synthesis are currently of the order of 108 distinct molecules. To put this in context, there are 1013 ways of arranging the 21 proteinogenic amino acids in chains up to 10 units long. Given that we know that a number of these compounds have potent biological activity, it would be highly desirable to be able to search them all to identify leads for new drug molecules. Large libraries of oligonucleotides can be synthesized combinatorially and translated into peptides using systems based on biological replication such as mRNA display, with selected molecules identified by DNA sequencing; but these methods are limited to BBs that are compatible with cellular machinery. In order to search the vast tracts of chemical space beyond nucleic acids and natural peptides, an alternative approach is required. DNA-templated synthesis (DTS) could enable us to meet this challenge. DTS controls chemical product formation by using the specificity of DNA hybridization to bring selected reactants into close proximity, and is capable of the programmed synthesis of many distinct products in the same reaction vessel. By making use of dynamic, programmable DNA processes, it is possible to engineer a system that can translate instructions coded as a sequence of DNA bases into a chemical structure-a process analogous to the action of the ribosome in living organisms but with the potential to create a much more chemically diverse set of products. It is also possible to ensure that each product molecule is tagged with its identifying DNA sequence. Compound libraries synthesized in this way can be exposed to selection against suitable targets, enriching successful molecules. The encoding DNA can then be amplified using the polymerase chain reaction and decoded by DNA sequencing. More importantly, the DNA instruction sequences can be mutated and reused during multiple rounds of amplification, translation, and selection. In other words, DTS could be used as the foundation for a system of synthetic molecular evolution, which could allow us to efficiently search a vast chemical space. This has huge potential to revolutionize materials discovery-imagine being able to evolve molecules for light harvesting, or catalysts for CO2 fixation. The field of DTS has developed to the point where a wide variety of reactions can be performed on a DNA template. Complex architectures and autonomous "DNA robots" have been implemented for the controlled assembly of BBs, and these mechanisms have in turn enabled the one-pot synthesis of large combinatorial libraries. Indeed, DTS libraries are being exploited by pharmaceutical companies and have already found their way into drug lead discovery programs. This Account explores the processes involved in DTS and highlights the challenges that remain in creating a general system for molecular discovery by evolution.

DNA T-junctions for studies of DNA origami assembly


KG Young, B Najafi, J Bath, AJ Turberfield

DNA-templated peptide assembly


J Jin, EG Baker, J Bath, DN Woolfson, AJ Turberfield

DNA origami dimensions and structure measured by solution X-ray scattering


MA Baker, AJ Tuckwell, JF Berengut, J Bath, F Benn, AP Duff, AE Whitten, KE Dunn, RM Hynson, AJ Turberfield, LK Lee

Quantitative single molecule surface-enhanced Raman scattering by optothermal tuning of DNA origami-assembled plasmonic nanoantennas

ACS Nano American Chemical Society 10 (2016) 9809-9815

S Simoncelli, E-M Roller, P Urban, R Schreiber, AJ Turberfield, T Liedl, T Lohmueller

DNA origami is a powerful approach for assembling plasmonic nanoparticle dimers and Raman dyes with high yields and excellent positioning control. Here we show how optothermal induced shrinking of a DNA origami template can be employed to control the gap sizes between two 40 nm gold nanoparticles in a range of 1 nm – 2 nm. The high field confinement achieved with this optothermal approach was demonstrated by detection of surface-enhanced Raman spectroscopy (SERS) signals from single molecules that are precisely placed within the DNA origami template that spans the nanoparticle gap. By comparing the SERS intensity with respect to the field enhancement in the plasmonic hotspot region, we found good agreement between measurement and theory. Our straightforward approach for the fabrication of addressable plasmonic nanosensors by DNA origami demonstrates a path toward future sensing applications with single-molecule resolution.

Ordering gold nanoparticles with DNA origami nanoflowers

ACS Nano American Chemical Society 10 (2016) 7303-7306

A Turberfield, R Schreiber, A Ardavan, I Santiago

Nanostructured materials, including plasmonic metamaterials made from gold and silver nanoparticles, provide access to new materials properties. The assembly of nanoparticles into extended arrays can be controlled through surface functionalization and the use of increasingly sophisticated linkers. We present a versatile way to control the bonding symmetry of gold nanoparticles by wrapping them in flower-shaped DNA origami structures. These ‘nanoflowers’ assemble into two-dimensonal gold nanoparticle lattices with symmetries that can be controlled through auxiliary DNA linker strands. Nanoflower lattices are true composites: interactions between the gold nanoparticles are mediated entirely by DNA, and the DNA origami will only fold into its designed form in the presence of the gold nanoparticles.

The formal language and design principles of autonomous DNA walker circuits

ACS Synthetic Biology American Chemical Society 5 (2016) 878-884

M Boemo, AE Lucas, AJ Turberfield, L Cardelli

Simple computation can be performed using the interactions between single-stranded molecules of DNA. These interactions are typically toehold-mediated strand displacement reactions in a well-mixed solution. We demonstrate that a DNA circuit with tethered reactants is a distributed system and show how it can be described as a stochastic Petri net. The system can be verified by mapping the Petri net onto a continuous-time Markov chain, which can also be used to find an optimal design for the circuit. This theoretical machinery can be applied to create software that automatically designs a DNA circuit, linking an abstract propositional formula to a physical DNA computation system that is capable of evaluating it. We conclude by introducing example mechanisms that can implement such circuits experimentally and discuss their individual strengths and weaknesses.

An Autonomous Molecular Assembler for Programmable Chemical Synthesis.

Nature Chemistry Nature Publishing Group (2016)

Meng, RA Muscat, ML McKee, PJ Milnes, El-Sagheer, JN Bath, Davis, Brown, RK O'Reilly, Turberfield

Molecular machines that assemble polymers in a programmed sequence are fundamental to life. They are also an achievable goal of nanotechnology. Here, we report synthetic molecular machinery made from DNA which controls and records the formation of covalent bonds. We show that an autonomous cascade of DNA hybridization reactions can create oligomers, from building blocks linked by olefin or peptide bonds, with a sequence defined by a reconfigurable molecular program. The system can also be programmed to achieve combinatorial assembly. The sequence of assembly reactions, and thus the structure, of each oligomer synthesized is recorded in a DNA molecule which enables this information to be recovered by PCR amplification followed by DNA sequencing.