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.

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.

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.

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.

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.

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.