Jinglin Fu

Interenzyme Substrate Diffusion for an Enzyme Cascade Organized on Spatially Addressable DNA Nanostructures

Spatially addressable DNA nanostructures facilitate the self-assembly of heterogeneous elements with precisely controlled patterns. Here we organized discrete glucose oxidase (GOx)/horseradish peroxidase (HRP) enzyme pairs on specific DNA origami tiles with controlled interenzyme spacing and position. The distance between enzymes was systematically varied from 10 to 65 nm, and the corresponding activities were evaluated. The study revealed two different distance-dependent kinetic processes associated with the assembled enzyme pairs. Strongly enhanced activity was observed for those assemblies in which the enzymes were closely spaced, while the activity dropped dramatically for enzymes as little as 20 nm apart. Increasing the spacing further resulted in a much weaker distance dependence. Combined with diffusion modeling, the results suggest that Brownian diffusion of intermediates in solution governed the variations in activity for more distant enzyme pairs, while dimensionally limited diffusion of intermediates across connected protein surfaces contributed to the enhancement in activity for closely spaced GOx/HRP assemblies. To further test the role of limited dimensional diffusion along protein surfaces, a noncatalytic protein bridge was inserted between GOx and HRP to connect their hydration shells. This resulted in substantially enhanced activity of the enzyme pair.

Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm

Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.

A DNA tweezer-actuated enzyme nanoreactor

The functions of regulatory enzymes are essential to modulating cellular pathways. Here we report a tweezer-like DNA nanodevice to actuate the activity of an enzyme/cofactor pair. A dehydrogenase and NAD(+) cofactor are attached to different arms of the DNA tweezer structure and actuation of enzymatic function is achieved by switching the tweezers between open and closed states. The enzyme/cofactor pair is spatially separated in the open state with inhibited enzyme function, whereas in the closed state, enzyme is activated by the close proximity of the two molecules. The conformational state of the DNA tweezer is controlled by the addition of specific oligonucleotides that serve as the thermodynamic driver (fuel) to trigger the change. Using this approach, several cycles of externally controlled enzyme inhibition and activation are successfully demonstrated. This principle of responsive enzyme nanodevices may be used to regulate other types of enzymes and to introduce feedback or feed-forward control loops.

Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

Spatially-Interactive Biomolecular Networks Organized by Nucleic Acid Nanostructures

Living systems have evolved a variety of nanostructures to control the molecular interactions that mediate many functions including the recognition of targets by receptors, the binding of enzymes to substrates, and the regulation of enzymatic activity. Mimicking these structures outside of the cell requires methods that offer nanoscale control over the organization of individual network components. Advances in DNA nanotechnology have enabled the design and fabrication of sophisticated one-, two- and three-dimensional (1D, 2D, and 3D) nanostructures that utilize spontaneous and sequence-specific DNA hybridization. Compared with other self-assembling biopolymers, DNA nanostructures offer predictable and programmable interactions and surface features to which other nanoparticles and biomolecules can be precisely positioned. The ability to control the spatial arrangement of the components while constructing highly organized networks will lead to various applications of these systems. For example, DNA nanoarrays with surface displays of molecular probes can sense noncovalent hybridization interactions with DNA, RNA, and proteins and covalent chemical reactions. DNA nanostructures can also align external molecules into well-defined arrays, which may improve the resolution of many structural determination methods, such as X-ray diffraction, cryo-EM, NMR, and super-resolution fluorescence. Moreover, by constraint of target entities to specific conformations, self-assembled DNA nanostructures can serve as molecular rulers to evaluate conformation-dependent activities. This Account describes the most recent advances in the DNA nanostructure directed assembly of biomolecular networks and explores the possibility of applying this technology to other fields of study. Recently, several reports have demonstrated the DNA nanostructure directed assembly of spatially interactive biomolecular networks. For example, researchers have constructed synthetic multienzyme cascades by organizing the position of the components using DNA nanoscaffolds in vitro or by utilizing RNA matrices in vivo. These structures display enhanced efficiency compared with the corresponding unstructured enzyme mixtures. Such systems are designed to mimic cellular function, where substrate diffusion between enzymes is facilitated and reactions are catalyzed with high efficiency and specificity. In addition, researchers have assembled multiple choromophores into arrays using a DNA nanoscaffold that optimizes the relative distance between the dyes and their spatial organization. The resulting artificial light-harvesting system exhibits efficient cascading energy transfers. Finally, DNA nanostructures have been used as assembly templates to construct nanodevices that execute rationally designed behaviors, including cargo loading, transportation, and route control.

Microfluidic picoliter-scale translational spontaneous sample introduction for high-speed capillary electrophoresis.

A novel microfluidic picoliter-scale sample introduction approach was developed by combining the spontaneous injection approach with a capillary electrophoresis (CE) system based on a short capillary and slotted-vial array. A droplet splitting phenomenon at the capillary inlet end during the spontaneous sample introduction process was observed for the first time. On the basis of this phenomenon, a translational spontaneous injection approach was established to reduce sample injection volumes to the sub-100 pL range. A versatile high-speed capillary electrophoresis (HSCE) system was built on the basis of this sample injection approach with separation performance comparable to or even better than those reported in microfluidic chip-based CE systems. The HSCE system was composed of a short fused-silica capillary and an automated sample introduction system with slotted sample and buffer reservoirs and a computer-programmed translational stage. The translational spontaneous sample injection was performed by linearly moving the stage, allowing the capillary inlet first to enter the sample solution and then removing it. A droplet was left at the tip end and spontaneously drawn into the capillary by surface tension effect to achieve sample injection. The stage was continuously moved to allow the capillary inlet to be immersed into the buffer solution, and CE separation was performed by applying a high voltage between the buffer and waste reservoirs. With the use of the novel system, high-speed and efficient capillary zone electrophoresis (CZE) separation of a mixture of five fluorescein isothiocyanate (FITC) labeled amino acids was achieved within 5.4 s in a short capillary with a separation length of 15 mm, reaching separation efficiencies up to 0.40 microm plate height. Outstanding peak height precisions ranging from 1.2% to 3.7% RSD were achieved in 51 consecutive separations. By extension of the separation length to 50 mm, both high-speed and high-resolution CZE separation of eight FITC-labeled amino acids could be obtained in less than 21 s with theoretical plates ranging from 163,000 to 251,000 (corresponding to 0.31-0.20 microm plate heights). The present HSCE system also allowed fast chiral separations of FITC-labeled amino acids under micellar electrokinetic chromatography (MEKC) mode within 6.5 s.

Controlled drug release by a nanorobot

A tiny, locked box made of DNA opens up to release drug molecules in the presence of target cells.

Peptide-modified surfaces for enzyme immobilization

Background Chemistry and particularly enzymology at surfaces is a topic of rapidly growing interest, both in terms of its role in biological systems and its application in biocatalysis. Existing protein immobilization approaches, including noncovalent or covalent attachments to solid supports, have difficulties in controlling protein orientation, reducing nonspecific absorption and preventing protein denaturation. New strategies for enzyme immobilization are needed that allow the precise control over orientation and position and thereby provide optimized activity. Methodology/Principal Findings A method is presented for utilizing peptide ligands to immobilize enzymes on surfaces with improved enzyme activity and stability. The appropriate peptide ligands have been rapidly selected from high-density arrays and when desirable, the peptide sequences were further optimized by single-point variant screening to enhance both the affinity and activity of the bound enzyme. For proof of concept, the peptides that bound to β-galactosidase and optimized its activity were covalently attached to surfaces for the purpose of capturing target enzymes. Compared to conventional methods, enzymes immobilized on peptide-modified surfaces exhibited higher specific activity and stability, as well as controlled protein orientation. Conclusions/Significance A simple method for immobilizing enzymes through specific interactions with peptides anchored on surfaces has been developed. This approach will be applicable to the immobilization of a wide variety of enzymes on surfaces with optimized orientation, location and performance, and provides a potential mechanism for the patterned self-assembly of multiple enzymes on surfaces.

A Three-Enzyme Pathway with an Optimised Geometric Arrangement to Facilitate Substrate Transfer.

Cascade reactions drive and regulate a variety of metabolic activities. Efficient coupling of substrate transport between enzymes is important for overall pathway activity and also controls the depletion of intermediate molecules that drive the reaction forward. Here, we assembled a three‐enzyme pathway on a series of DNA nanoscaffolds to investigate the dependence of their activities on spatial arrangement. Unlike previous studies, the overall activity of the three‐enzyme pathway relied less on inter‐enzyme distance and more on the geometric patterns that arranged them within a relatively small range of 10–30 nm. Pathway intermediate detection demonstrated that the assembled enzyme systems quickly depleted the intermediate molecules through efficient reaction coupling.

Exploring peptide space for enzyme modulators

A method is presented for screening high-density arrays to discover peptides that bind and modulate enzyme activity. A polyvinyl alcohol solution was applied to array surfaces to limit the diffusion of product molecules released from enzymatic reactions, allowing the simultaneous measurement of enzyme activity and binding at each peptide spot. For proof of concept, it was possible to identify peptides that bound to horseradish peroxidase, alkaline phosphatase, and beta-galactosidase and substantially altered enzyme activity by comparing the binding level of peptide to enzyme and bound enzyme activity. This basic technique may be generally applicable to find peptides or other small molecules that modify enzyme activity.