A Robust and Efficient Method to Purify DNA-Scaffolded Nanostructures by Gravity-Driven Size Exclusion Chromatography.

In recent decades, nucleic acid self-assemblies have emerged as popular nanomaterials due to their programmable and robust assembly, prescribed geometry, and versatile functionality. However, it remains a challenge to purify large quantities of DNA nanostructures or DNA-templated nanocomplexes for various applications. Commonly used purification methods are either limited by a small scale or incompatible with functionalized structures. To address this unmet need, we present a robust and scalable method of purifying DNA nanostructures by Sepharose resin-based size exclusion. The resin column can be manually packed in-house with reusability. The separation is driven by a low-pressure gravity flow in which large DNA nanostructures are eluted first followed by smaller impurities of ssDNA and proteins. We demonstrated the efficiency of the method for purifying DNA origami assemblies and protein-immobilized DNA nanostructures. Compared to routine agarose gel electrophoresis that yields 1 µg or less of purified products, this method can purify ∼100-1000 µg of DNA nanostructures in less than 30 min, with the overall collection yield of 50-70% of crude preparation mixture. The purified nanocomplexes showed more precise activity in evaluating enzyme functions and antibody-triggered activation of complement protein reactions.

Modulation of Enzyme Cascade Activity by Local Substrate Enrichment and Exclusion on DNA Nanostructures.

Substrate confinement and channeling play a critical role in multienzyme pathways and are considered to impact the catalytic efficiency and specificity of biomimetic and artificial nanoreactors. Here we reported a modulation of a multienzyme system with the cascade activity impacted by the surface affinity binding to substrate molecules. A DNA origami modified with aptamers was used to bind and enrich ATP molecules in the local area of immobilized enzymes, thereby enhancing the activity of an enzyme cascade by more than 2-fold. Alternatively, DNA nanostructure modified with blocked aptamers does not bind with ATP, thereby reducing the activity of the enzyme cascade. The Michaelis-Menten kinetics showed decreased apparent KM values (∼3-fold lower) for enzyme nanostructures modified with aptamers, suggesting the higher effective substrate concentration near enzymes due to the local enrichment of substrates. Conversely, increased apparent KM values (∼2-fold higher) were observed for enzyme nanostructures modified with blocked aptamers, possibly due to the exclusion of substrates approaching the surface. The similar concept of this modified surface-substrate interaction should be applicable to other multienzyme systems immobilized on nanostructures, which could be useful in the development of biomimetic nanoreactors.

Biomimetic Compartments Scaffolded by Nucleic Acid Nanostructures.

The behaviors of living cells are governed by a series of regulated and confined biochemical reactions. The design and successful construction of synthetic cellular reactors can be useful in a broad range of applications that will bring significant scientific and economic impact. Over the past few decades, DNA self-assembly has enabled the design and fabrication of sophisticated 1D, 2D, and 3D nanostructures, and is applied to organizing a variety of biomolecular components into prescribed 2D and 3D patterns. In this Concept, the recent and exciting progress in DNA-scaffolded compartmentalizations and their applications in enzyme encapsulation, lipid membrane assembly, artificial transmembrane nanopores, and smart drug delivery are in focus. Taking advantage of these features promises to deliver breakthroughs toward the attainment of new synthetic and biomimetic reactors.

2D Enzyme Cascade Network with Efficient Substrate Channeling by Swinging Arms.

In living cells, compartmentalized or membrane-associated enzymes are often assembled into large networks to cooperatively catalyze cascade reaction pathways essential for cellular metabolism. Here, we report the assembly of an artificial 2D enzyme network of two cascade enzymes-glucose-6-phosphate dehydrogenase (G6PDH) and lactate dehydrogenase (LDH)-on a wireframe DNA origami template. Swinging arms were used to facilitate the transport of the redox intermediate of NAD+ /NADH between enzyme pairs on the array. The assemblies of 2D enzyme networks were characterized by gel electrophoresis and visualized by atomic force microscopy (AFM). The spatial arrangements of multiple enzyme pairs were optimized to facilitate efficient substrate channeling by exploiting the programmability of DNA origami to manipulate the key parameters of swinging arm length and stoichiometry. Compared with a single enzyme pair, the 2D organized enzyme systems exhibited higher reaction efficiency due to the promoted transfer of intermediates within the network.

An activity transition from NADH dehydrogenase to NADH oxidase during protein denaturation.

A decrease in the specific activity of an enzyme is commonly observed when the enzyme is inappropriately handled or is stored over an extended period. Here, we reported a functional transition of an FMN-bound diaphorase (FMN-DI) that happened during the long-term storage process. It was found that FMN-DI did not simply lose its ß-nicotinamide adenine diphosphate (NADH) dehydrogenase activity after a long-time storage, but obtained a new enzyme activity of NADH oxidase. Further mechanistic studies suggested that the alteration of the binding strength of an FMN cofactor with a DI protein could be responsible for this functional switch of the enzyme.

DNA-Mediated Proximity-Based Assembly Circuit for Actuation of Biochemical Reactions.

Smart nanodevices that integrate molecular recognition and signal production hold great promise for the point-of-care (POC) diagnostic applications. Herein, the development of a DNA-mediated proximity assembly of biochemical reactions, which was capable of sensing various bio-targets and reporting easy-to-read signals is reported. The circuit was composed of a DNA hairpin-locked catalytic cofactor with inhibited activity. Specific molecular inputs can trigger a conformational switch of the DNA locks through the mechanisms of toehold displacement and aptamer switching, exposing an active cofactor. The subsequent assembly of an enzyme/cofactor pair actuated a reaction to produce colorimetric or fluorescence signals for detecting target molecules. The developed system could be potentially applied to smart biosensing in molecular diagnostics and POC tests.

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.

Improved high-speed capillary electrophoresis system using a short capillary and picoliter-scale translational spontaneous injection.

Here, we describe an improved high-speed CE (HSCE) system using a short capillary and translational spontaneous sample injection. Several important factors for consideration in system design as well as various factors influencing the performance of the HSCE system were investigated in detail. The performance of this HSCE system was demonstrated in electrophoretic separation of FITC-labeled amino acids. Under optimized conditions, baseline separation of eight amino acids and FITC were achieved in 21 s with the plate heights ranging from 0.20 to 0.31 µm, corresponding to a separation rate up to 20 700 theoretical plates per second. The separation speed and efficiency of the optimized high-speed CE system are comparable to or even better than those reported in microchip-based CE systems.

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.

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.

Reduction of Promiscuous Peptides-Enzyme Inhibition and Aggregation by Negatively Charged Biopolymers.

In this work, peptides selected from a microarray were found to inhibit ß-gal with promiscuous mechanisms. Peptides inhibited the enzyme in a noncompetitive kinetics, and the inhibition of enzyme activities was reduced under high enzyme concentrations and the addition of detergent. Dynamic light scattering and atomic force microscope revealed that peptide/enzyme aggregation was related to inhibited enzyme activities. Positively charged residues of arginine and lysine were critical for the enzyme inhibition. The preincubation of peptide inhibitors with negatively charged biopolymers of polyphosphates, ssDNA, and low pI peptides could increase the residual activity of peptide-inhibited enzyme, possibly due to the disruption of the electrostatic interaction between positively charged peptide residues and the ß-gal surface. Further, negative biopolymers were able to recover the activity of the aggregated peptide/ß-gal complex. Negatively charged biopolymers could be used in high-throughput screening assays to reduce peptides/protein aggregation and thereby minimize promiscuous inhibitions.

DNA Nanoscaffolds for Multienzyme Systems Assembly.

Multienzyme reactions play an important role in cellular metabolic functions. The assembly of a metabolon is often observed, in which the position and the orientation of composite enzymes are optimized to facilitate the substrate transport. The recent progress of DNA nanotechnology is promising to organize the assembly of bimolecular complexes with precise controlled geometric patterns at nanoscale, such as enzyme cascades assembly, biomimetic substrate channeling, and compartmentalization. Here, we present detailed protocols of using DNA nanoscaffolds to assemble a multienzyme system with control over spatial interactions and arrangements of individual components. The protocols include the preparation and purification of DNA nanostructures, the bioconjugation of DNA with proteins and cofactors, the chromatography purification of DNA-conjugated biomolecules, the characterization of assemblies by routine gel electrophoresis and advanced AFM imaging, as well as the activity evaluation of multienzyme assemblies.

Inactivation Kinetics of G-Quadruplex/Hemin Complex and Optimization for More Reliable Catalysis.

Reliable catalysis is critical for the synthesis of various chemicals, molecular sensing and biomedicine. G-quadruplex/Hemin (GQH) complex, a peroxidase-mimicking DNAzyme, has been widely used in various publications. However, a concern exists about the unstable kinetics of GQH-catalyzed peroxidation. This work investigates several factors that result in the inactivation of GQH and the signal degradation during long reaction periods, including pH, buffer component, the selection of substrate and the oxidation damage of cofactor. Using colorimetric and fluorescent assays, GQH was found to be highly unstable under basic conditions with 50 % of GQH activity lost within 2 minutes at high H2 O2 concentrations. Appropriate conditions and substrates are suggested for accurately characterizing GQH-catalyzed reactions, as well as optimization to improve the catalytic reliability, such as the use of polyhistidine and cascade reactions. These results could be useful for GQH-related applications.

Rapid Nucleic Acid Reaction Circuits for Point-of-care Diagnosis of Diseases.

An urgent need exists for a rapid, cost-effective, facile, and reliable nucleic acid assay for mass screening to control and prevent the spread of emerging pandemic diseases. This urgent need is not fully met by current diagnostic tools. In this review, we summarize the current state-of-the-art research in novel nucleic acid amplification and detection that could be applied to point-of-care (POC) diagnosis and mass screening of diseases. The critical technological breakthroughs will be discussed for their advantages and disadvantages. Finally, we will discuss the future challenges of developing nucleic acid-based POC diagnosis.

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.

DNA-Scaffolded Proximity Assembly and Confinement of Multienzyme Reactions.

Cellular functions rely on a series of organized and regulated multienzyme cascade reactions. The catalytic efficiencies of these cascades depend on the precise spatial organization of the constituent enzymes, which is optimized to facilitate substrate transport and regulate activities. Mimicry of this organization in a non-living, artificial system would be very useful in a broad range of applications-with impacts on both the scientific community and society at large. Self-assembled DNA nanostructures are promising applications to organize biomolecular components into prescribed, multidimensional patterns. In this review, we focus on recent progress in the field of DNA-scaffolded assembly and confinement of multienzyme reactions. DNA self-assembly is exploited to build spatially organized multienzyme cascades with control over their relative distance, substrate diffusion paths, compartmentalization and activity actuation. The combination of addressable DNA assembly and multienzyme cascades can deliver breakthroughs toward the engineering of novel synthetic and biomimetic reactors.

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.

Constructing Submonolayer DNA Origami Scaffold on Gold Electrode for Wiring of Redox Enzymatic Cascade Pathways.

Advances in biomimetic microelectronics offer a range of patterned assemblies of proteins and cells for in vitro metabolic engineering where coordinated biochemical pathways allow cell metabolism to be characterized and potentially controlled on a chip. To achieve these goals, developing new methods for interfacing biological systems to microelectronic devices has been in urgent demand. Here, we report the assembly of a DNA origami-templated enzymatic cascade (glucose oxidase and horseradish peroxidase) on gold electrodes, where a monolayer of DNA origami is anchored on gold electrodes via Au-S chemistry, to create programmable, electrochemically driven biomimetic device containing both biochemical and electronic components. Upon the posing of a specific electrical potential, substrates/products flow through the enzyme pair and the end product transfers electrons to the electrode. The steady state flux of the distance-dependent enzymatic cascade reactions is translated into a steady state current signal that records the overall enzyme activity. This biological system can be finely tuned by varying the distance between the enzyme pair, which opens new routes to interface microelectronic devices to biological functions.

Self-Assembly of DNA-Minocycline Complexes by Metal Ions with Controlled Drug Release.

Here we reported a study of metal ions-assisted assembly of DNA-minocycline (MC) complexes and their potential application for controlling MC release. In the presence of divalent cations of magnesium or calcium ions (M2+), MC, a zwitterionic tetracycline analogue, was found to bind to phosphate groups of nucleic acids via an electrostatic bridge of phosphate (DNA)-M2+-MC. We investigated multiple parameters for affecting the formation of DNA-Mg2+-MC complex, including metal ion concentrations, base composition, DNA length, and single- versus double-stranded DNA. For different nitrogen bases, single-stranded poly(A)20 and poly(T)20 showed a higher MC entrapment efficiency of DNA-Mg2+-MC complex than poly(C)20 and poly(G)20. Single-stranded DNA was also found to form a more stable DNA-Mg2+-MC complex than double-stranded DNA. Between different divalent metal ions, we observed that the formation of DNA-Ca2+-MC complex was more stable and efficient than the formation of DNA-Mg2+-MC complex. Toward drug release, we used agarose gel to encapsulate DNA-Mg2+-MC complexes and monitored MC release. Some DNA-Mg2+-MC complexes could prolong MC release from agarose gel to more than 10 days as compared with the quick release of free MC from agarose gel in less than 1 day. The released MC from DNA-Mg2+-MC complexes retained the anti-inflammatory bioactivity to inhibit nitric oxide production from pro-inflammatory macrophages. The reported study of metal ion-assisted DNA-MC assembly not only increased our understanding of biochemical interactions between tetracycline molecules and nucleic acids but also contributed to the development of a highly tunable drug delivery system to mediate MC release for clinical applications.

Microarray Selection of Cooperative Peptides for Modulating Enzyme Activities.

Recently, peptide microarrays have been used to distinguish proteins, antibodies, viruses, and bacteria based on their binding to random sequence peptides. We reported on the use of peptide arrays to identify enzyme modulators that involve screening an array of 10,000 defined and addressable peptides on a microarray. Primary peptides were first selected to inhibit the enzyme at low µM concentrations. Then, new peptides were found to only bind strongly with the enzyme-inhibitor complex, but not the native enzyme. These new peptides served as secondary inhibitors that enhanced the inhibition of the enzyme together with the primary peptides. Without the primary peptides, the secondary effect peptides had little effect on the enzyme activity. Conversely, we also selected peptides that recovered the activities of inhibited enzyme-peptide complex. The selection of cooperative peptide pairs will provide a versatile toolkit for modulating enzyme functions, which may potentially be applied to drug discovery and biocatalysis.