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Super-selective multivalent ligand-receptor interactions display a signature step-like onset in binding when meeting a characteristic density of target receptors. Materials engineered for super-selective binding generally display a high number of flexible ligands to enhance the systems' avidity. In many biological processes, however, ligands are present in moderate copy numbers and arranged in spatio-temporal patterns. In this low-valency regime, the rigidity of the ligand-presenting architecture plays a critical role in the selectivity of the multivalent complex through decrease of the entropic penalty of binding. Exploiting the precision in spatial design inherent to the DNA nanotechnology, we engineered a library of rigid architectures to explore how valency, affinity, and nano-spacing control the presence of super-selectivity in multivalent binding. A micromolar monovalent affinity was required for super-selective binding to be observed within low-valency systems, and the transition point for stable interactions was measured at hexavalent ligand presentation, setting the limits of the low valency regime. Super-selective binding was observed for all hexavalent architectures, and, more strikingly, the ligand pattern determined the selectivity onset. Hereby, we demonstrate for the first time that nano-control of geometric patterns can be used to discriminate between receptor densities in a super-selective manner. Materials that were indistinguishable in their molecular composition and ligand valency bound with various efficacies on surfaces with constant receptor densities. We define this new phenomenon in super-selective binding as multivalent pattern recognition.

The kinetics of DNA hybridization are fundamental to biological processes and DNA-based technologies. However, the precise physical mechanisms that determine why different DNA sequences hybridize at different rates are not well understood. Secondary structure is one predictable factor that influences hybridization rates but is not sufficient on its own to fully explain the observed sequence-dependent variance. In this context, we measured hybridization rates of 43 different DNA sequences that are not predicted to form secondary structure and present a parsimonious physically justified model to quantify our observations. Accounting only for the combinatorics of complementary nucleating interactions and their sequence-dependent stability, the model achieves good correlation with experiment with only two free parameters. Our results indicate that greater repetition of Watson–Crick pairs increases the number of initial states able to proceed to full hybridization, with the stability of those pairings dictating the likelihood of such progression, thus providing new insight into the physical factors underpinning DNA hybridization rates.

DNA is an attractive candidate for integration into nanoelectronics as a biological nanowire due to its linear geometry, definable base sequence, easy, inexpensive and non-toxic replication and self-assembling properties. Recently we discovered that by intercalating Ag+ in polycytosine-mismatch oligonucleotides, the resulting C-Ag+-C duplexes are able to conduct charge efficiently. To map the functionality and biostability of this system, we built and characterized internally-functionalized DNA nanowires through non-canonical, Ag+-mediated base pairing in duplexes containing cytosine-cytosine mismatches. We assessed the thermal and chemical stability of ion-coordinated duplexes in aqueous solutions and conclude that the C-Ag+-C bond forms DNA duplexes with replicable geometry, predictable thermodynamics, and tunable length. We demonstrated continuous ion chain formation in oligonucleotides of 11–50 nucleotides (nt), and enzyme ligation of mixed strands up to six times that length. This construction is feasible without detectable silver nanocluster contaminants. Functional gene parts for the synthesis of DNA- and RNA-based, C-Ag+-C duplexes in a cell-free system have been constructed in an Escherichia coli expression plasmid and added to the open-source BioBrick Registry, paving the way to realizing the promise of inexpensive industrial production. With appropriate design constraints, this conductive variant of DNA demonstrates promise for use in synthetic biological constructs as a dynamic nucleic acid component and contributes molecular electronic functionality to DNA that is not already found in nature. We propose a viable route to fabricating stable DNA nanowires in cell-free and synthetic biological systems for the production of self-assembling nanoelectronic architectures.

Studying the self-assembly behavior of DNA origami allows a better understanding of molecular assembly characteristics at the nanoscale. Presently, the mechanisms governing growth and dynamics of DNA origami assembly are still not very clear and there is a lack of direct visualization of the growth processes on the long single-strand scaffold. Here, we investigate the kinetics, especially the real-time seeding growth process of six special designs of 2D DNA origami at room temperature (RT) without the assistance of denaturing chemicals. The prealignment of single-strand long scaffold and logical seeding growth behaviors are revealed during the growth process at RT. Furthermore, we studied the thermal stability of the DNA nanostructures under limited structural defects. Revealed characteristics of seeding growth can be used to build large and complex DNA nanodevices capable of performing logical operations with nanometer precision.

Biology demonstrates how a near infinite array of complex systems and structures at many scales can originate from the self-assembly of component parts on the nanoscale. But to fully exploit the benefits of self-assembly for nanotechnology, a crucial challenge remains: How do we rationally encode well-defined global architectures in subunits that are much smaller than their assemblies? Strain accumulation via geometric frustration is one mechanism that has been used to explain the self-assembly of global architectures in diverse and complex systems a posteriori. Here we take the next step and use strain accumulation as a rational design principle to control the length distributions of self-assembling polymers. We use the DNA origami method to design and synthesize a molecular subunit known as the PolyBrick, which perturbs its shape in response to local interactions via flexible allosteric blocking domains. These perturbations accumulate at the ends of polymers during growth, until the deformation becomes incompatible with further extension. We demonstrate that the key thermodynamic factors for controlling length distributions are the intersubunit binding free energy and the fundamental strain free energy, both which can be rationally encoded in a PolyBrick subunit. While passive polymerization yields geometrical distributions, which have the highest statistical length uncertainty for a given mean, the PolyBrick yields polymers that approach Gaussian length distributions whose variance is entirely determined by the strain free energy. We also show how strain accumulation can in principle yield length distributions that become tighter with increasing subunit affinity and approach distributions with uniform polymer lengths. Finally, coarse-grained molecular dynamics and Monte Carlo simulations delineate and quantify the dominant forces influencing strain accumulation in a molecular system. This study constitutes a fundamental investigation of the use of strain accumulation as a rational design principle in molecular self-assembly.

Since its introduction in the early 2000s, DNA origami had a big impact on the development of nanotechnology by gathering numerous applications. During this time, many tools were designed and used to generate arbitrary shapes capable of self-assembly which make this technique more approachable. In this paper, we have created the map of Romania at nanoscale dimensions by using a new open-source software - PERDIX. For this purpose, we used a scaffold strand with a length of 6959 nucleotides and 162 staple strands with a variable length ranging between 20 and 63 nucleotides. All the computational tools that were used in this experiment are open-source and user-friendly.

This study reports the first experimental evidence of using DNA as a polymeric additive to enhance protein crystallization. Using three kinds of DNA with different molecular weights&mdash, calf DNA, salmon DNA, and herring DNA&mdash, this study showed an improvement in the success rate of lysozyme crystallization, as compared to control experiments, especially at low lysozyme concentration. The improvement of crystallization is particularly significant in the presence of calf DNA with the highest molecular weight. Calf DNA also speeds up the induction time of lysozyme crystallization and increases the number of crystals per drop. We hypothesized the effect of DNA on protein crystallization may be due to the combination of excluded volume effect, change of water&rsquo, s surface tension, and the water competition effect. This work confirms predications of the potential use of DNA as a polymeric additive to enhance protein crystallization, potentially applied to systems with limited protein available or difficult to crystallize.

In this communication, we show that active enzymes can be delivered into HEK293 cells in vitro when they are attached to tubular DNA origami nanostructures. We use bioluminescent enzymes as a cargo and monitor their activity from a cell lysate. The results show that the enzymes stay intact and retain their activity in the transfection process. The method is highly modular, which makes it a compelling candidate for a great variety of delivery applications.

The nuclear pore complex (NPC) is the gatekeeper for nuclear transport in eukaryotic cells. A key component of the NPC is the central shaft lined with intrinsically disordered proteins (IDPs) known as FG-Nups, which control the selective molecular traffic. Here, we present an approach to realize artificial NPC mimics that allows controlling the type and copy number of FG-Nups. We constructed 34 nm-wide 3D DNA origami rings and attached different numbers of NSP1, a model yeast FG-Nup, or NSP1-S, a hydrophilic mutant. Using (cryo) electron microscopy, we find that NSP1 forms denser cohesive networks inside the ring compared to NSP1-S. Consistent with this, the measured ionic conductance is lower for NSP1 than for NSP1-S. Molecular dynamics simulations reveal spatially varying protein densities and conductances in good agreement with the experiments. Our technique provides an experimental platform for deciphering the collective behavior of IDPs with full control of their type and position., FG-Nups are disordered proteins in the nuclear pore complex (NPC) where they selectively control nuclear transport. Here authors build NPC-mimics based on DNA origami rings which attach a certain numbers of Nups to analyse those nanopores by cryoEM and conductance measurements.

We present a detailed coarse-grained computer simulation and single molecule fluorescence study of the walking dynamics and mechanism of a DNA bipedal motor striding on a DNA origami. In particular, we study the dependency of the walking efficiency and stepping kinetics on step size. The simulations accurately capture and explain three different experimental observations. These include a description of the maximum possible step size, a decrease in the walking efficiency over short distances and a dependency of the efficiency on the walking direction with respect to the origami track. The former two observations were not expected and are non-trivial. Based on this study, we suggest three design modifications to improve future DNA walkers. Our study demonstrates the ability of the oxDNA model to resolve the dynamics of complex DNA machines, and its usefulness as an engineering tool for the design of DNA machines that operate in the three spatial dimensions.

1700692 Fully addressable DNA nanostructures, especially DNA origami, possess huge potential to serve as inherently biocompatible and versatile molecular platforms. However, their use as delivery vehicles in therapeutics is compromised by their low stability and poor transfection rates. This study shows that DNA origami can be coated by precisely defined one-to-one protein-dendron conjugates to tackle the aforementioned issues. The dendron part of the conjugate serves as a cationic binding domain that attaches to the negatively charged DNA origami surface via electrostatic interactions. The protein is attached to dendron through cysteine-maleimide bond, making the modular approach highly versatile. This work demonstrates the coating using two different proteins: bovine serum albumin (BSA) and class II hydrophobin (HFBI). The results reveal that BSA-coating significantly improves the origami stability against endonucleases (DNase I) and enhances the transfection into human embryonic kidney (HEK293) cells. Importantly,it is observed that BSA-coating attenuates the activation of immune response in mouse primary splenocytes. Serum albumin is the most abundant protein in the blood with a long circulation half-life and has already found clinically approved applications in drug delivery. It is therefore envisioned that the proposed system can open up further opportunities to tune the properties of DNA nanostructures in biological environment, and enable their use in various delivery applications.

We present a modelling framework, and basic model parameterization, for the study of DNA origami folding at the level of DNA domains. Our approach is explicitly kinetic and does not assume a specific folding pathway. The binding of each staple is associated with a free-energy change that depends on staple sequence, the possibility of coaxial stacking with neighbouring domains, and the entropic cost of constraining the scaffold by inserting staple crossovers. A rigorous thermodynamic model is difficult to implement as a result of the complex, multiply connected geometry of the scaffold: we present a solution to this problem for planar origami. Coaxial stacking of helices and entropic terms, particularly when loop closure exponents are taken to be larger than those for ideal chains, introduce interactions between staples. These cooperative interactions lead to the prediction of sharp assembly transitions with notable hysteresis that are consistent with experimental observations. We show that the model reproduces the experimentally observed consequences of reducing staple concentration, accelerated cooling, and absent staples. We also present a simpler methodology that gives consistent results and can be used to study a wider range of systems including non-planar origami.

A versatile procedure to build high-symmetry objects and to calculate their corresponding small-angle scattering intensity is presented. Starting from a set of vertex positions, available from a large and extensible database, it is possible to build several types of bodies using spherical subunits. A fast implementation, based on the Debye formula using a histogram of distance, is then used to compute the theoretical scattering intensity. Since the model is built from the definition of a small set of parameters, it is possible to perform an optimization of structural parameters against experimental data. Finally, affine size polydispersities can be easily included by the rescaling of the histogram of the positions used in the calculations. Several examples of the calculations are presented, demonstrating the method and its applicability.

Highlights • We review recent advances in nucleic acid chemistry and polymerase engineering that have enabled the synthesis, replication, and evolution of a wide range of nucleic acid-like synthetic genetic polymers (XNAs) with improved chemical and biological stability. • We discuss the likely biotechnological impact of the further development of XNA technology for the generation of novel ligands, enzymes, and nanostructures with tailor-made chemistry., Nucleic acids display remarkable properties beyond information storage and propagation. The well-understood base pairing rules have enabled nucleic acids to be assembled into nanostructures of ever increasing complexity. Although nanostructures can be constructed using other building blocks, including peptides and lipids, it is the capacity to evolve that sets nucleic acids apart from all other nanoscale building materials. Nonetheless, the poor chemical and biological stability of DNA and RNA constrain their applications. Recent advances in nucleic acid chemistry and polymerase engineering enable the synthesis, replication, and evolution of a range of synthetic genetic polymers (XNAs) with improved chemical and biological stability. We discuss the impact of this technology on the generation of XNA ligands, enzymes, and nanostructures with tailor-made chemistry.