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.

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.

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-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.

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.

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.

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.

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.

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.

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.

DNA-crowded enzyme complexes with enhanced activities and stabilities.

We present a robust and simple method to prepare DNA-crowded enzyme complexes by directly assembling long DNA duplexes on the enzyme surface. DNA-crowded enzyme complexes show boosted substrate turnover numbers, and increased stabilities against various storage conditions. They could be potentially scaled up for applications in biomaterials and biotechnology.

Computational and experimental analysis of short peptide motifs for enzyme inhibition.

The metabolism of living systems involves many enzymes that play key roles as catalysts and are essential to biological function. Searching ligands with the ability to modulate enzyme activities is central to diagnosis and therapeutics. Peptides represent a promising class of potential enzyme modulators due to the large chemical diversity, and well-established methods for library synthesis. Peptides and their derivatives are found to play critical roles in modulating enzymes and mediating cellular uptakes, which are increasingly valuable in therapeutics. We present a methodology that uses molecular dynamics (MD) and point-variant screening to identify short peptide motifs that are critical for inhibiting ß-galactosidase (ß-Gal). MD was used to simulate the conformations of peptides and to suggest short motifs that were most populated in simulated conformations. The function of the simulated motifs was further validated by the experimental point-variant screening as critical segments for inhibiting the enzyme. Based on the validated motifs, we eventually identified a 7-mer short peptide for inhibiting an enzyme with low µM IC50. The advantage of our methodology is the relatively simplified simulation that is informative enough to identify the critical sequence of a peptide inhibitor, with a precision comparable to truncation and alanine scanning experiments. Our combined experimental and computational approach does not rely on a detailed understanding of mechanistic and structural details. The MD simulation suggests the populated motifs that are consistent with the results of the experimental alanine and truncation scanning. This approach appears to be applicable to both natural and artificial peptides. With more discovered short motifs in the future, they could be exploited for modulating biocatalysis, and developing new medicine.

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.

Chemical shift assignments of Ribosomal protein S1 from Mycobacterium tuberculosis.

RpsA, also known as ribosomal protein S1, is an essential protein required for translation initiation of mRNAs when their Shine-Dalgarno sequence is degenerated (Sorensen et al. 1998). In addition, RpsA of Mycobacterium tuberculosis (M. tb) is involved in trans-translation, which is an effective system mediated by tmRNA-SmpB to release stalled ribosomes from mRNA in the presence of rare codons (Keiler 2008). Shi et al. found that POA binds to RpsA of Mtb and disrupts the formation of RpsA-tmRNA complex (Shi et al. 2011) and mutations at the C-terminus of RpsA confer PZA resistance. The previous work reported the pyrazinoic acid-binding domain of RpsA (Yang et al. Mol Microbiol 95:791-803, 2015). However, the HSQC spectra of the isolated S1 domain does not overlap with that of MtRpsA280-438, suggesting that substantial interactions occur between the flexible C-terminus and the S1 domain in MtRpsA .To further study the PZA resistance and how substantial interactions influence/affect protein structure, using heteronuclear NMR spectroscopy, we have completed backbone and side-chain 1H, 15N, 13C chemical shift assignments of MtRpsA280-438 which contains S1 domain and the flexible C-terminus. These NMR resonance assignments provide the framework for detailed characterization of the solution-state protein structure determination, dynamic studies of this domain, as well as NMR-based drug discovery efforts.

Spatial Organization of Enzyme Cascade on a DNA Origami Nanostructure.

Self-assembled DNA nanostructures hold great promise to organize multi-enzyme systems with the precise control of the geometric arrangements. Enzymes modified with single-stranded DNA anchors are assembled onto the DNA origami tiles by hybridizing with the corresponding complementary strands displayed on the surface of the DNA nanostructures. Here, we describe a protocol of assembling a two-enzyme cascade on a discrete, rectangular DNA origami tile, where the distance between enzymes is precisely controlled for investigating the distance-dependent cascade activities.

Self-assembled Nucleic Acid Nanostructures for Cancer Theranostic Medicines.

Theranostic medicine has become more promising in cancer treatment, where the cancer diagnosis and chemotherapy are combined for early diagnosis and precise treatment with improved efficacy and reduced side effects. Nanotechnology has played a critical role in developing various nanomaterials with engendered smart functions and targeted delivery. The rapid development of structural DNA nanotechnology has enabled the design and fabrication of complex nanostructures with prescribed 1D, 2D and 3D patterns in vitro and in vivo. Self-assembled DNA nanostructures can serve as drug delivery platforms that are integrated with various functions ranging from molecular recognition and computations, dynamically structural switch to carrying molecular payloads and selectively release. In this review, we summarize recent exciting progress of using DNA nanostructures to engineer novel smart drug-delivery systems potential for treating cancer.