Designing Rigid DNA Origami Templates for Molecular Visualization Using Cryo-EM.

DNA origami, a method for constructing nanostructures from DNA, offers potential for diverse scientific and technological applications due to its ability to integrate various molecular functionalities in a programmable manner. In this study, we examined the impact of internal crossover distribution and the compositional uniformity of staple strands on the structure of multilayer DNA origami using cryogenic electron microscopy (cryo-EM) single-particle analysis. A refined DNA object was utilized as an alignment framework in a host-guest model, where we successfully resolved an 8 kDa thrombin binding aptamer (TBA) linked to the host object.

Triple-Stranded DNA As a Structural Element in DNA Origami.

Molecular self-assembly with DNA origami offers an attractive route to fabricate arbitrary three-dimensional nanostructures. In DNA origami, B-form double-helical DNA domains (dsDNA) are commonly linked with covalent phosphodiester strand crossovers to build up three-dimensional objects. To expand the palette of structural motifs in DNA origami, here we describe hybrid duplex-triplex DNA motifs as pH-dependent building blocks in DNA origami.

A DNA origami rotary ratchet motor.

To impart directionality to the motions of a molecular mechanism, one must overcome the random thermal forces that are ubiquitous on such small scales and in liquid solution at ambient temperature. In equilibrium without energy supply, directional motion cannot be sustained without violating the laws of thermodynamics. Under conditions away from thermodynamic equilibrium, directional motion may be achieved within the framework of Brownian ratchets, which are diffusive mechanisms that have broken inversion symmetry.

Phage-free production of artificial ssDNA with Escherichia coli.

Artificial single-stranded DNA (ssDNA) with user-defined sequences and lengths up to the kilobase range is increasingly needed in mass quantities to realize the potential of emerging technologies such as genome editing and DNA origami. However, currently available biotechnological approaches for mass-producing ssDNA require dedicated, and thus costly, fermentation infrastructure, because of the risk of cross-contaminating manufacturer plants with self-replicating phages. Here we overcome this problem with an efficient, scalable, and cross-contamination-free method for the phage-free biotechnological production of artificial ssDNA with Escherichia coli.

A nanoscale reciprocating rotary mechanism with coordinated mobility control.

Biological molecular motors transform chemical energy into mechanical work by coupling cyclic catalytic reactions to large-scale structural transitions. Mechanical deformation can be surprisingly efficient in realizing such coupling, as demonstrated by the FF ATP synthase. Here, we describe a synthetic molecular mechanism that transforms a rotary motion of an asymmetric camshaft into reciprocating large-scale transitions in a surrounding stator orchestrated by mechanical deformation.

A synthetic tubular molecular transport system.

Creating artificial macromolecular transport systems that can support the movement of molecules along defined routes is a key goal of nanotechnology. Here, we report the bottom-up construction of a macromolecular transport system in which molecular pistons diffusively move through micrometer-long, hollow filaments. The pistons can cover micrometer distances in fractions of seconds.

Cryo-Electron Microscopy and Mass Analysis of Oligolysine-Coated DNA Nanostructures.

Cationic coatings can enhance the stability of synthetic DNA objects in low ionic strength environments such as physiological fluids. Here, we used single-particle cryo-electron microscopy (cryo-EM), pseudoatomic model fitting, and single-molecule mass photometry to study oligolysine and polyethylene glycol (PEG)-oligolysine-coated multilayer DNA origami objects. The coatings preserve coarse structural features well on a resolution of multiple nanometers but can also induce deformations such as twisting and bending.

Revealing the structures of megadalton-scale DNA complexes with nucleotide resolution.

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.

Monitoring polysorbate hydrolysis in biopharmaceuticals using a QC-ready free fatty acid quantification method.

Hydrolysis of the non-ionic surfactant polysorbate upon long-term storage poses significant challenges to development of biopharmaceutical liquid formulations. Low concentrations of intact surfactant may compromise its protective properties and thus affect protein stability. In addition, accumulation of polysorbate hydrolysis products is increasingly put into context with the formation of visible and subvisible particulates based on the low solubility of the main degradation products.

Biotechnological mass production of DNA origami.

DNA nanotechnology, in particular DNA origami, enables the bottom-up self-assembly of micrometre-scale, three-dimensional structures with nanometre-precise features. These structures are customizable in that they can be site-specifically functionalized or constructed to exhibit machine-like or logic-gating behaviour. Their use has been limited to applications that require only small amounts of material (of the order of micrograms), owing to the limitations of current production methods.