Sturdier DNA nanotubes via ligation

DNA nanotubes are crystalline self-assemblies of DNA tiles ~10 nm in diameter that readily grow tens of micrometers in length. Easy assembly, programmability, and stiffness make them interesting for many applications, but DNA nanotubes begin to melt at temperatures below 40 °C, break open when deposited on mica or scanned by AFM, and disintegrate in deionized water. These weaknesses can be traced to the presence of discontinuities in the phosphate backbone, called nicks. The nanotubes studied here have five nicks, one in the core of a tile and one at each corner. We report the successful ligation of all four corner nicks by T4 DNA ligase. Although ligation does not change the nanotubes’ stiffness, ligated nanotubes withstand temperatures over 70 °C, resist breaking during AFM, and are stable in pure water for over a month. Ligated DNA nanotubes are thus physically and chemically sturdy enough to withstand the manipulations necessary for many technological applications

Programmable molecular recognition based on the geometry of DNA nanostructures

From ligand–receptor binding to DNA hybridization, molecular recognition plays a central role in biology. Over the past several decades, chemists have successfully reproduced the exquisite specificity of biomolecular interactions. However, engineering multiple specific interactions in synthetic systems remains difficult. DNA retains its position as the best medium with which to create orthogonal, isoenergetic interactions, based on the complementarity of Watson–Crick binding. Here we show that DNA can be used to create diverse bonds using an entirely different principle: the geometric arrangement of blunt-end stacking interactions. We show that both binary codes and shape complementarity can serve as a basis for such stacking bonds, and explore their specificity, thermodynamics and binding rules. Orthogonal stacking bonds were used to connect five distinct DNA origami. This work, which demonstrates how a single attractive interaction can be developed to create diverse bonds, may guide strategies for molecular recognition in systems beyond DNA nanostructures

Properties of DNA- and Protein-Scaffolded Lipid Nanodiscs

The properties of natural lipid bilayers are vital to the regulation of many membrane proteins. Scaffolded nanodiscs provide an in vitro lipid bilayer platform to host membrane proteins in an environment that approximates native lipid bilayers. However, the properties of scaffold-enclosed bilayers may depart significantly from those of bulk cellular membranes. Therefore, to improve the usefulness of nanodiscs it is essential to understand the properties of lipids restricted by scaffolds. We used computational molecular dynamics and modeling approaches to understand the effects of nanodisc size, scaffold type (DNA or protein), and hydrophobic modification of DNA scaffolds on bilayer stability and degree to which the properties of enclosed bilayers approximate bulk bilayers. With respect to achieving bulk bilayer behavior, we found that charge neutralization of DNA scaffolds was more important than the total hydrophobic content of their modifications: bilayer properties were better for scaffolds having a large number of short alkyl chains than those having fewer long alkyl chains. Further, complete charge neutralization of DNA scaffolds enabled better lipid binding, and more stable bilayers, as shown by steered molecular dynamics simulations that measured the force required to dislodge scaffolds from lipid bilayer patches. Considered together, our simulations provide a guide to the design of DNA-scaffolded nanodiscs suitable for studying membrane proteins

Design and characterization of programmable DNA nanotubes

DNA self-assembly provides a programmable bottom-up approach for the synthesis of complex structures from nanoscale components. Although nanotubes are a fundamental form encountered in tile-based DNA self-assembly, the factors governing tube structure remain poorly understood. Here we report and characterize a new type of nanotube made from DNA double-crossover molecules (DAE-E tiles). Unmodified tubes range from 7 to 20 nm in diameter (4 to 10 tiles in circumference), grow as long as 50 μm with a persistence length of ~4 μm, and can be programmed to display a variety of patterns. A survey of modifications (1) confirms the importance of sticky-end stacking, (2) confirms the identity of the inside and outside faces of the tubes, and (3) identifies features of the tiles that profoundly affect the size and morphology of the tubes. Supported by these results, nanotube structure is explained by a simple model based on the geometry and energetics of B-form DNA

A population-based temporal logic gate for timing and recording chemical events

Engineered bacterial sensors have potential applications in human health monitoring, environmental chemical detection, and materials biosynthesis. While such bacterial devices have long been engineered to differentiate between combinations of inputs, their potential to process signal timing and duration has been overlooked. In this work, we present a two‐input temporal logic gate that can sense and record the order of the inputs, the timing between inputs, and the duration of input pulses. Our temporal logic gate design relies on unidirectional DNA recombination mediated by bacteriophage integrases to detect and encode sequences of input events. For an E. coli strain engineered to contain our temporal logic gate, we compare predictions of Markov model simulations with laboratory measurements of final population distributions for both step and pulse inputs. Although single cells were engineered to have digital outputs, stochastic noise created heterogeneous single‐cell responses that translated into analog population responses. Furthermore, when single‐cell genetic states were aggregated into population‐level distributions, these distributions contained unique information not encoded in individual cells. Thus, final differentiated sub‐populations could be used to deduce order, timing, and duration of transient chemical events