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. Our system utilizes a designed phagemid and an optimized helper plasmid. The phagemid encodes one gene of the M13 phage genome and a freely chosen custom target sequence, while the helper plasmid encodes the other genes of the M13 phage. The phagemid particles produced with this method are not capable of self-replication in the absence of the helper plasmid. This enables cross-contamination-free biotechnological production of ssDNA at any contract manufacturer. Furthermore, we optimized the process parameters to reduce by-products and increased the maximal product concentration up to 83 mg L-1 of ssDNA in a stirred-tank bioreactor, thus realizing up to a 40-fold increase in maximal product concentration over previous scalable phage-free ssDNA production methods.

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 symmetry1-5. Ratcheting is thought to underpin the function of many natural biological motors, such as the F1F0-ATPase6-8, and it has been demonstrated experimentally in synthetic microscale systems (for example, to our knowledge, first in ref. 3) and also in artificial molecular motors created by organic chemical synthesis9-12. DNA nanotechnology13 has yielded a variety of nanoscale mechanisms, including pivots, hinges, crank sliders and rotary systems14-17, which can adopt different configurations, for example, triggered by strand-displacement reactions18,19 or by changing environmental parameters such as pH, ionic strength, temperature, external fields and by coupling their motions to those of natural motor proteins20-26. This previous work and considering low-Reynolds-number dynamics and inherent stochasticity27,28 led us to develop a nanoscale rotary motor built from DNA origami that is driven by ratcheting and whose mechanical capabilities approach those of biological motors such as F1F0-ATPase.

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 F1FO 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. We design the mechanism using DNA origami, characterize its structure via cryo-electron microscopy, and examine its dynamic behavior using single-particle fluorescence microscopy and molecular dynamics simulations. While the camshaft can rotate inside the stator by diffusion, the stator's mechanics makes the camshaft pause at preferred orientations. By changing the stator's mechanical stiffness, we accelerate or suppress the Brownian rotation, demonstrating an allosteric coupling between the camshaft and the stator. Our mechanism provides a framework for manufacturing artificial nanomachines that function because of coordinated movements of their components.

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. We build the system using multi-layer DNA origami and analyze the structures of the components using transmission electron microscopy. We study the motion of the pistons along the tubes using single-molecule fluorescence microscopy and perform Langevin simulations to reveal details of the free energy surface that directs the motions of the pistons. The tubular transport system achieves diffusivities and displacement ranges known from natural molecular motors and realizes mobility improvements over five orders of magnitude compared to previous artificial random walker designs. Electric fields can also be employed to actively pull the pistons along the filaments, thereby realizing a nanoscale electric rail system. Our system presents a platform for artificial motors that move autonomously driven by chemical fuels and for performing nanotribology studies, and it could form a basis for future molecular transportation networks.

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. Higher-density coatings also led to internal structural deformations in the DNA origami test objects, in which a designed honeycomb-type helical lattice was deformed into a more square-lattice-like pattern. Under physiological ionic strength, where the uncoated objects disassembled, the coated objects remained intact but they shrunk in the helical direction and expanded in the direction perpendicular to the helical axis. Helical details like major/minor grooves and crossover locations were not discernible in cryo-EM maps that we determined of DNA origami coated with oligolysine and PEG-oligolysine, whereas these features were visible in cryo-EM maps determined from the uncoated reference objects. Blunt-ended double-helical interfaces remained accessible underneath the coating and may be used for the formation of multimeric DNA origami assemblies that rely on stacking interactions between blunt-ended helices. The ionic strength requirements for forming multimers from coated DNA origami differed from those needed for uncoated objects. Using single-molecule mass photometry, we found that the mass of coated DNA origami objects prior to and after incubation in low ionic strength physiological conditions remained unchanged. This finding indicated that the coating effectively prevented strand dissociation but also that the coating itself remained stable in place. Our results validate oligolysine coatings as a powerful stabilization method for DNA origami but also reveal several potential points of failure that experimenters should watch to avoid working with false premises.

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. 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.

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. Despite of this potential negative impact on product quality, quantification of the released free fatty acids is performed commonly in an indirect and consequently insensitive manner by determining the remaining PS content or by cumbersome methods, which are unsuitable for routine testing in quality control laboratories. For this purpose, this study describes the development and qualification of a label-free, reliable liquid-chromatography single quad mass detector (LC-QDa)-based method capable of resolving slight changes in the free fatty acid profile which can be readily integrated into quality control facilities. The practical utility of the herein described method is outlined by a case study on the real-time storage stability of a formulated monoclonal antibody.

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. But many proposed applications, for example as therapeutic agents or in complex materials, could be realized if more material could be used. In DNA origami, a nanostructure is assembled from a very long single-stranded scaffold molecule held in place by many short single-stranded staple oligonucleotides. Only the bacteriophage-derived scaffold molecules are amenable to scalable and efficient mass production; the shorter staple strands are obtained through costly solid-phase synthesis or enzymatic processes. Here we show that single strands of DNA of virtually arbitrary length and with virtually arbitrary sequences can be produced in a scalable and cost-efficient manner by using bacteriophages to generate single-stranded precursor DNA that contains target strand sequences interleaved with self-excising 'cassettes', with each cassette comprising two Zn2+-dependent DNA-cleaving DNA enzymes. We produce all of the necessary single strands of DNA for several DNA origami using shaker-flask cultures, and demonstrate end-to-end production of macroscopic amounts of a DNA origami nanorod in a litre-scale stirred-tank bioreactor. Our method is compatible with existing DNA origami design frameworks and retains the modularity and addressability of DNA origami objects that are necessary for implementing custom modifications using functional groups. With all of the production and purification steps amenable to scaling, we expect that our method will expand the scope of DNA nanotechnology in many areas of science and technology.