A Light-Powered Single-Stranded DNA Molecular Motor with Colour-Selective Single-Step Control.

Top-down control of small motion is possible through top-down controlled molecular motors in replacement of larger actuators like MEMS or NEMS (micro- or nano-electromechanical systems) in the current precision technology. Improving top-down control of molecular motors to every single step is desirable for this purpose, and also for synchronization of motor actions for amplified effects. Here we report a designed single-stranded DNA molecular motor powered by alternated ultraviolet and visible light for processive track-walking, with the two light colours each locking the motor in a full directional step to allow saturated driving but no overstepping. This novel nano-optomechanical driving mechanism pushes the top-down control of molecular motors down to every single step, thus providing a key technical capability to advance the molecular motor-based precision technology and also motor synchronization for amplified effects.

Reproducible Preparation of Proteopolymersomes via Sequential Polymer Film Hydration and Membrane Protein Reconstitution.

Film rehydration method is commonly used for membrane protein (MP) reconstitution into block copolymer (BCP), but the lack of control in the rehydration step formed a heterogeneous population of proteopolymersomes that interferes with the characterization and performance of devices incorporating them. To improve the self-assembly of polymersomes with simultaneous MP reconstitution, the study reported herein aimed to understand the effects of different variants of the rehydration procedure on the MP reconstitution into BCP membranes. The model MP used in this study was AquaporinZ (AqpZ), an α-helical MP that has been shown to have a high permeation rate exclusive to water molecules. Comparing four rehydration methods differing in the hydration time (i.e., brief wetting or full hydration) and medium (i.e., in buffer or AqpZ stock solution), prehydration with buffer prior to adding AqpZ was found to be most desirable and reproducible reconstitution method because it gave rise to the highest proportion of well-formed vesicles with intact AqpZ functionality as evidenced by the transmission electron microscopy images, dynamic light scattering, and stopped-flow analyses. The mechanisms by which effective AqpZ reconstitution takes place were also investigated and discussed. Small-angle X-ray scattering analysis shows that hydrating the initially dry multilamellar BCP films allows the separation of lamellae. This is anticipated to increase the membrane fluidity that facilitates a fast and spontaneous integration of AqpZ as the detergent concentration is considerably lowered below its critical micelle concentration. Dilution of detergent can result in precipitation of proteins in the absence of well-fluidized membranes for protein integration that underscores the importance of membrane fluidity in MP reconstitution.

Controlled Supramolecular Self-Assembly of Super-charged ß-Lactoglobulin A-PEG Conjugates into Nanocapsules.

The synthesis and characterization of a new protein-polymer conjugate composed of ß lactoglobulin A (ßLG A) and poly(ethylene glycol) PEG is described. ßLG A was selectively modified to self-assemble by super-charging via amination or succinylation followed by conjugation with PEG. An equimolar mixture of the oppositely charged protein-polymer conjugates self-assemble into spherical capsules of 80-100 nm in diameter. The self-assembly proceeds by taking simultaneous advantage of the amphiphilicity and polyelectrolyte nature of the protein-polymer conjugate. These protein-polymer capsules or proteinosomes are reminiscent of protein capsids, and are capable of encapsulating solutes in their interior. We envisage this approach to be applicable to other globular proteins.

Photo-induced conjugation of tetrazoles to modified and native proteins.

Bio-orthogonal chemistry has been widely used for conjugation of polymer molecules to proteins. Here, we demonstrate the conjugation of polyethylene glycol (PEG) to bovine beta-lactoglobulin (BLG) by photo-induced cyclo-addition of tetrazole-appended PEG and allyl-modified BLG. During the course of the investigation, a significant side-reaction was found to occur for the conjugation of PEG-tetrazole to native BLG. Further exploration of the underlying chemistry reveals that the presence of a tryptophan residue is sufficient for conjugation of tetrazole-modified molecules.

A light-operated integrated DNA walker-origami system beyond bridge burning.

Integrating rationally designed DNA molecular walkers and DNA origami platforms is a promising route towards advanced nano-robotics of diverse functions. Unleashing the full potential in this direction requires DNA walker-origami systems beyond the present simplistic bridge-burning designs for automated repeatable operation and scalable nano-robotic functions. Here we report such a DNA walker-origami system integrating an advanced light-powered DNA bipedal walker and a ∼170 nm-long rod-like DNA origami platform. This light-powered walker is fully qualified as a genuine translational molecular motor, and relies entirely on pure mechanical effects that are complicated by the origami surface but must be preserved for the walker's proper operation. This is made possible by tailor-designing the origami for optimal match with the walker to best preserve its core mechanics. A new fluorescence method is combined with site-controlled motility experiments to yield distinct and reliable signals for the walker's self-directed and processive motion despite origami-complicated fluorophore emission. The resultant integrated DNA walker-origami system provides a 'seed' system for future development of advanced light-powered DNA nano-robots (e.g., for scalable walker-automated chemical synthesis), and also truly bio-mimicking nano-muscles powered by genuine artificial translational molecular motors.

Autonomous DNA molecular motor tailor-designed to navigate DNA origami surface for fast complex motion and advanced nanorobotics.

Nanorobots powered by designed DNA molecular motors on DNA origami platforms are vigorously pursued but still short of fully autonomous and sustainable operation, as the reported systems rely on manually operated or autonomous but bridge-burning molecular motors. Expanding DNA nanorobotics requires origami-based autonomous non-bridge-burning motors, but such advanced artificial molecular motors are rare, and their integration with DNA origami remains a challenge. Here, we report an autonomous non-bridge-burning DNA motor tailor-designed for a triangle DNA origami substrate. This is a translational bipedal molecular motor but demonstrates effective translocation on both straight and curved segments of a self-closed circular track on the origami, including sharp ~90° turns by a single hand-over-hand step. The motor is highly directional and attains a record-high speed among the autonomous artificial molecular motors reported to date. The resultant DNA motor-origami system, with its complex translational-rotational motion and big nanorobotic capacity, potentially offers a self-contained "seed" nanorobotic platform to automate or scale up many applications.

An intercompartmental enzymatic cascade reaction in channel-equipped polymersome-in-polymersome architectures.

Compartmentalization, as a design principle, is a prerequisite for the functioning of eukaryotic cells. Although cell mimics in the form of single vesicular compartments such as liposomes or polymersomes have been tremendously successful, investigations of the corresponding higher-order architectures, in particular bilayer-based multicompartment vesicles, have only recently gained attention. We hereby demonstrate a multicompartment cell-mimetic nanocontainer, built-up from fully synthetic membranes, which features an inner compartment equipped with a channel protein and a semi-permeable outer compartment that allows passive diffusion of small molecules. The functionality of this multicompartment architecture is demonstrated by a cascade reaction between enzymes that are segregated in separate compartments. The unique architecture of polymersomes, which combines stability with a cell-membrane-mimetic environment, and their assembly into higher-order architectures could serve as a design principle for new generation drug-delivery vehicles, biosensors, and protocell models.