DNA origami has attracted substantial attention since its invention ten years ago, due to the seemingly infinite possibilities that it affords for creating customized nanoscale objects. Although the basic concept of DNA origami is easy to understand, using custom DNA origami in practical applications requires detailed know-how for designing and producing the particles with sufficient quality and for preparing them at appropriate concentrations with the necessary degree of purity in custom environments. Such know-how is not readily available for newcomers to the field, thus slowing down the rate at which new applications outside the field of DNA nanotechnology may emerge. To foster faster progress, we share in this article the experience in making and preparing DNA origami that we have accumulated over recent years. We discuss design solutions for creating advanced structural motifs including corners and various types of hinges that expand the design space for the more rigid multilayer DNA origami and provide guidelines for preventing undesired aggregation and on how to induce specific oligomerization of multiple DNA origami building blocks. In addition, we provide detailed protocols and discuss the expected results for five key methods that allow efficient and damage-free preparation of DNA origami. These methods are agarose-gel purification, filtration through molecular cut-off membranes, PEG precipitation, size-exclusion chromatography, and ultracentrifugation-based sedimentation. The guide for creating advanced design motifs and the detailed protocols with their experimental characterization that we describe here should lower the barrier for researchers to accomplish the full DNA origami production workflow.

Many strategies have been pursued to trap and monitor single proteins over time in order to detect the molecular mechanisms of these essential nanomachines. Single protein sensing with nanopores is particularly attractive because it allows label-free high-bandwidth detection based on ion currents.Here we present the Nanopore Electro-Osmotic trap (NEOtrap) that allows trapping and observing single proteins for hours with sub-millisecond time resolution. The NEOtrap is formed by docking a DNA-origami sphere onto a passivated solid-state nanopore, which seals off a nanocavity of a userdefined size and creates an electro-osmotic flow that traps nearby particles irrespective of their charge. We demonstrate the NEOtrap's ability to sensitively distinguish proteins based on size and shape, and discriminate nucleotide-dependent protein conformations, as exemplified by the chaperone protein Hsp90. Given the experimental simplicity and capacity for label-free single-protein detection over the broad bio-relevant time range, the NEOtrap opens new avenues to study the molecular kinetics underlying protein function..

Molecular traffic across lipid membranes is a vital process in cell biology that involves specialized biological pores with a great variety of pore diameters, from fractions of a nanometer to >30 nm. Creating artificial membrane pores covering similar size and complexity will aid the understanding of transmembrane molecular transport in cells, while artificial pores are also a necessary ingredient for synthetic cells. Here, we report the construction of DNA origami nanopores that have an inner diameter as large as 30 nm. We developed methods to successfully insert these ultrawide pores into the lipid membrane of giant unilamellar vesicles (GUVs) by administering the pores concomitantly with vesicle formation in an inverted-emulsion cDICE technique. The reconstituted pores permit the transmembrane diffusion of large macromolecules, such as folded proteins, which demonstrates the formation of large membrane-spanning open pores. The pores are size selective, as dextran molecules with a diameter up to 28 nm can traverse the pores, whereas larger dextran molecules are blocked. By FRAP measurements and modeling of the GFP influx rate, we find that up to hundreds of pores can be functionally reconstituted into a single GUV. Our technique bears great potential for applications across different fields from biomimetics, to synthetic biology, to drug delivery.

The methods of DNA nanotechnology enable the rational design of custom shapes that self-assemble in solution from sets of DNA molecules. DNA origami, in which a long template DNA single strand is folded by many short DNA oligonucleotides, can be employed to make objects comprising hundreds of unique DNA strands and thousands of base pairs, thus in principle providing many degrees of freedom for modelling complex objects of defined 3D shapes and sizes. Here, we address the problem of accurate structural validation of DNA objects in solution with cryo-EM based methodologies. By taking into account structural fluctuations, we can determine structures with improved detail compared to previous work. To interpret the experimental cryo-EM maps, we present molecular-dynamics-based methods for building pseudo-atomic models in a semi-automated fashion. Among other features, our data allows discerning details such as helical grooves, single-strand versus double-strand crossovers, backbone phosphate positions, and single-strand breaks. Obtaining this higher level of detail is a step forward that now allows designers to inspect and refine their designs with base-pair level interventions.