A proximity effect has been invoked to explain the enhanced activity of enzyme cascades on DNA scaffolds. Using the cascade reaction carried out by glucose oxidase and horseradish peroxidase as a model system, here we study the kinetics of the cascade reaction when the enzymes are free in solution, when they are conjugated to each other and when a competing enzyme is present. No proximity effect is found, which is in agreement with models predicting that the rapidly diffusing hydrogen peroxide intermediate is well mixed. We suggest that the reason for the activity enhancement of enzymes localized by DNA scaffolds is that the pH near the surface of the negatively charged DNA nanostructures is lower than that in the bulk solution, creating a more optimal pH environment for the anchored enzymes. Our findings challenge the notion of a proximity effect and provide new insights into the role of DNA scaffolds.

Molecular shuttles have been built from motor proteins capable of moving cargo along engineered paths. We illustrate alternative methods of controlling the direction of motion of microtubules on engineered kinesin tracks, how to load cargo covalently to microtubules, and how to exploit UV-induced release of caged ATP combined with enzymatic ATP degradation by hexokinase to turn the shuttles on and off sequentially. These are the first steps in the development of a tool kit to utilize molecular motors for the construction of nanoscale assembly lines.One of the central challenges in nanotechnology is how to assemble nanoscale building blocks including molecular wires and nanoscale switches into addressable devices, where the building blocks can be either synthesized, self-assembled in solution, or fabricated at the nanoscale. Ideally, a nanoscale conveyor belt would allow us to transport a selected nanoscale item over a defined distance and then facilitate the docking under defined angles with a second item of choice. One labor-intensive approach is to use tips of scanning probe microscopes to pick up, move, and release atoms, single molecules, and nanoscale objects in a controlled manner, one at a time. 1 Nanoscale objects created in this way are therefore pieces of art rather than devices of economical value. Methods need to be developed that allow the parallel assembly of nanoscale building blocks in large numbers. Here we combine methods of micro-and nanofabrication, with molecular assembly, and molecular motors powered chemically by adenosine triphosphate (ATP) to construct molecular shuttles that move under user control. This represents the first step in learning how to construct and operate nanoscale conveyor belts in large numbers.Key features of conveyor belts are that they (a) are driven by a force-generating motor, (b) transport cargo unidirectionally between well-defined positions, (c) accommodate loading and docking of cargo, and (d) can be externally switched on and off. These features are independent of the length scale at which conveyor belts are constructed and operated. If operation at the nanoscale is desired, molecular motors are required to generate force to propel cargo. Furthermore, schemes have to be developed to guide molecular movement along precisely controlled tracks, to load and unload cargo, and to control the speed of motion noninvasively. We will review and describe novel approaches that can serve as universal elements of a tool kit to construct and operate molecular shuttles. Molecular Motors. Nature has evolved unique molecules, termed motor proteins, that transport cargo over long distances or rotate loads in an energy-dependent manner. The characteristics of these motor proteins are far superior to all demonstrated miniaturized synthetic engines. 2 Motor proteins generate more force, have better fuel efficiency, and are smaller in size than any man-made device.

The concept of "metabolic channeling" as a result of rapid transfer of freely diffusing intermediate substrates between two enzymes on nanoscale scaffolds is examined using simulations and mathematical models. The increase in direct substrate transfer due to the proximity of the two enzymes provides an initial but temporary boost to the throughput of the cascade and loses importance as product molecules of enzyme 1 (substrate molecules of enzyme 2) accumulate in the surrounding container. The characteristic time scale at which this boost is significant is given by the ratio of container volume to the product of substrate diffusion constant and interenzyme distance and is on the order of milliseconds to seconds in some experimental systems. However, the attachment of a large number of enzyme pairs to a scaffold provides an increased number of local "targets", extending the characteristic time. If substrate molecules for enzyme 2 are sequestered by an alternative reaction in the container, a scaffold can result in a permanent boost to cascade throughput with a magnitude given by the ratio of the above-defined time scale to the lifetime of the substrate molecule in the container. Finally, a weak attractive interaction between substrate molecules and the scaffold creates a "virtual compartment" and substantially accelerates initial throughput. If intermediate substrates can diffuse freely, placing individual enzyme pairs on scaffolds is only beneficial in large cells, unconfined extracellular spaces or in systems with sequestering reactions.

Enzymatic catalysis is essential to cell survival. In many instances, enzymes that participate in reaction cascades have been shown to assemble into metabolons in response to the presence of the substrate for the first enzyme. However, what triggers metabolon formation has remained an open question. Through a combination of theory and experiments, we show that enzymes in a cascade can assemble via chemotaxis. We apply microfluidic and fluorescent spectroscopy techniques to study the coordinated movement of the first four enzymes of the glycolysis cascade: hexokinase, phosphoglucose isomerase, phosphofructokinase and aldolase. We show that each enzyme independently follows its own specific substrate gradient, which in turn is produced by the preceding enzymatic reaction. Furthermore, we find that the chemotactic assembly of enzymes occurs even under cytosolic crowding conditions.

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Cells regulate active transport of intracellular cargo using motor proteins. Recent nanobiotechnology efforts aim to adapt motor proteins to power the movement and assembly of synthetic materials. A motor-protein-based nanoscale transport system (molecular shuttle) requires that the motion of the shuttles be guided along tracks. This study investigates the principles by which microtubules, serving as shuttle units, are guided along micrometer-scale kinesin-coated chemical and topographical tracks, where the efficiency of guidance is determined by events at the track boundary. Thus, we measure the probability of guiding as microtubules reach the track boundary of (1) a chemical edge between kinesin-coated and kinesin-free surfaces, (2) a topography-only wall coated completely with kinesin, and (3) a kinesin-free wall next to a kinesin-coated bottom surface (topography and chemistry combined). We present a guiding mechanism for each surface type that takes into account the physical properties of microtubule filaments and the surface properties (geometry, chemistry), and elucidate the contributions of surface topography and chemistry. Our experimental and theoretical results show that track edges that combine both topography and chemistry guide microtubules most frequently (approximately 90% of all events). By applying the principles of microtubule guidance by microfabricated surfaces, one may design and build motor-protein-powered devices optimized for transport.

Biosensors can be miniaturized by either injecting smaller volumes into micro- and nanofluidic devices or immersing increasingly sophisticated particles known as 'smart dust' into the sample. The term 'smart dust' originally referred to cubic-millimetre wireless semiconducting sensor devices that could invisibly monitor the environment in buildings and public spaces, but later it also came to include functional micrometre-sized porous silicon particles used to monitor yet smaller environments. The principal challenge in designing smart dust biosensors is integrating transport functions with energy supply into the device. Here, we report a hybrid microdevice that is powered by ATP and relies on antibody-functionalized microtubules and kinesin motors to transport the target analyte into a detection region. The transport step replaces the wash step in traditional double-antibody sandwich assays. Owing to their small size and autonomous function, we envision that large numbers of such smart dust biosensors could be inserted into organisms or distributed into the environment for remote sensing.

Biological systems have evolved to harness non-equilibrium processes from the molecular to the macro scale. It is currently a grand challenge of chemistry, materials science, and engineering to understand and mimic biological systems that have the ability to autonomously sense stimuli, process these inputs, and respond by performing mechanical work. New chemical systems are responding to the challenge and form the basis for future responsive, adaptive, and active materials. In this article, we describe a particular biochemical-biomechanical network based on the microtubule cytoskeletal filament – itself a non-equilibrium chemical system. We trace the non-equilibrium aspects of the system from molecules to networks and describe how the cell uses this system to perform active work in essential processes. Finally, we discuss how microtubule-based engineered systems can serve as testbeds for autonomous chemical robots composed of biological and synthetic components.