A DNA Origami-Based Gene Editing System for Efficient Gene Therapy in Vivo

DNA nanostructures have played an important role in the development of novel drug delivery systems. Herein, we report a DNA origami-based CRISPR/Cas9 gene editing system for efficient gene therapy in vivo. In our design, a PAM-rich region precisely organized on the surface of DNA origami can easily recruit and load sgRNA/Cas9 complex by PAM-guided assembly and pre-designed DNA/RNA hybridization. After loading the sgRNA/Cas9 complex, the DNA origami can be further rolled up by the locking strands with a disulfide bond. With the incorporation of DNA aptamer and influenza hemagglutinin (HA) peptide, the cargo-loaded DNA origami can realize the targeted delivery and effective endosomal escape. After reduction by GSH, the opened DNA origami can release the sgRNA/Cas9 complex by RNase H cleavage to achieve a pronounced gene editing of a tumor-associated gene for gene therapy in vivo. This rationally developed DNA origami-based gene editing system presents a new avenue for the development of gene therapy.     

Supramolecular Polyphenol-DNA Microparticles for In Vivo Adjuvant and Antigen Co-Delivery and Immune Stimulation

DNA-based materials have attracted interest due to the tunable structure and encoded biological functionality of nucleic acids. A simple and general approach to synthesize DNA-based materials with fine control over morphology and bioactivity is important to expand their applications. Here, we report the synthesis of DNA-based particles via the supramolecular assembly of tannic acid (TA) and DNA. Uniform particles with different morphologies are obtained using a variety of DNA building blocks. The particles enable the co-delivery of cytosine-guanine adjuvant sequences and the antigen ovalbumin in model cells. Intramuscular injection of the particles in mice induces antigen-specific antibody production and T cell responses with no apparent toxicity. Protein expression in cells is shown using capsules assembled from TA and plasmid DNA. This work highlights the potential of TA as a universal material for directing the supramolecular assembly of DNA into gene and vaccine delivery platforms.     

Photo-Controlled Gating of Selective Bacterial Membrane Interaction and Enhanced Antibacterial Activity for Wound Healing

Reversible biointerfaces are essential for on-demand molecular recognition to regulate stimuli-responsive bioactivity such as specific interactions with cell membranes. The reversibility on a single platform allows the smart material to kill pathogens or attach/detach cells. Herein, we introduce a 2D-MoS₂ functionalized with cationic azobenzene that interacts selectively with either Gram-positive or Gram-negative bacteria in a light-gated fashion. The trans conformation ( trans -Azo-MoS₂ ) selectively kills Gram-negative bacteria, whereas the cis form ( cis -Azo-MoS₂ ), under UV light, exhibits antibacterial activity against Gram-positive strains. The mechanistic investigation indicates that the cis -Azo-MoS₂ exhibits higher affinity towards the membrane of Gram-positive bacteria compared to trans -Azo-MoS₂ . In case of Gram-negative bacteria, trans -Azo-MoS₂ internalizes more efficiently than cis -Azo-MoS₂ and generates intracellular ROS to kill the bacteria. While the trans -Azo-MoS₂ exhibits strong electrostatic interactions and internalizes faster into Gram-negative bacterial cells, cis -Azo-MoS₂ primarily interacts with Gram-positive bacteria through hydrophobic and H-bonding interactions. The difference in molecular mechanism leads to photo-controlled Gram-selectivity and enhanced antibacterial activity. We found strain-specific and high bactericidal activity (minimal bactericidal concentration, 0.65 μg/ml) with low cytotoxicity, which we extended to wound healing applications. This methodology provides a single platform for efficiently switching between conformers to reversibly control the strain-selective bactericidal activity regulated by light.     

Dynamic Hydrogen-Bonded Zinc Adeninate Framework (ZAF) for Immobilization of Catalytic DNA

Effective immobilization and delivery of genetic materials is at the forefront of biological and medical research directed toward tackling scientific challenges such as gene therapy and cancer treatment. Herein we present a biologically inspired hydrogen-bonded zinc adeninate framework (ZAF) consisting of zinc adeninate macrocycles that self-assemble into a 3D framework through adenine-adenine interactions. ZAF can efficiently immobilize DNAzyme with full protection against enzyme degradation and physiological conditions until it is successfully delivered into the nucleus. As compared to zeolitic imidazolate frameworks (ZIFs), ZAFs are twofold more biocompatible with a significant loading efficiency of 96 %. Overall, our design paves the way for expanding functional hydrogen-bonding-based systems as potential platforms for the loading and delivery of biologics.     

Programmable 3D Hexagonal Geometry of DNA Tensegrity Triangles

Non-canonical interactions in DNA remain under-explored in DNA nanotechnology. Recently, many structures with non-canonical motifs have been discovered, notably a hexagonal arrangement of typically rhombohedral DNA tensegrity triangles that forms through non-canonical sticky end interactions. Here, we find a series of mechanisms to program a hexagonal arrangement using: the sticky end sequence; triangle edge torsional stress; and crystallization condition. We showcase cross-talking between Watson–Crick and non-canonical sticky ends in which the ratio between the two dictates segregation by crystal forms or combination into composite crystals. Finally, we develop a method for reconfiguring the long-range geometry of formed crystals from rhombohedral to hexagonal and vice versa. These data demonstrate fine control over non-canonical motifs and their topological self-assembly. This will vastly increase the programmability, functionality, and versatility of rationally designed DNA constructs.     

A Spatially Programmable DNA Nanorobot Arm to Modulate Anisotropic Gold Nanoparticle Assembly by Enzymatic Excision

Programmable assembly of gold nanoparticle superstructures with precise spatial arrangement has drawn much attention for their unique characteristics in plasmonics and biomedicine. Bio-inspired methods have already provided programmable, molecular approaches to direct AuNP assemblies using biopolymers. The existing methods, however, predominantly use DNA as scaffolds to directly guide the AuNP interactions to produce intended superstructures. New paradigms for regulating AuNP assembly will greatly enrich the toolbox for DNA-directed AuNP manipulation and fabrication. Here, we developed a strategy of using a spatially programmable enzymatic nanorobot arm to modulate anisotropic DNA surface modifications and assembly of AuNPs. Through spatial controls of the proximity of the reactants, the locations of the modifications were precisely regulated. We demonstrated the control of the modifications on a single 15 nm AuNP, as well as on a rectangular DNA origami platform, to direct unique anisotropic AuNP assemblies. This method adds an alternative enzymatic manipulation to DNA-directed AuNP superstructure assembly.     

Aminopeptidase N-Responsive Conjugates with Tunable Charge-Reversal Properties for Highly Efficient Tumor Accumulation and Penetration

Tumor enzyme-responsive charge-reversal carriers can induce efficient transcytosis and lead to efficient tumor infiltration and potent anticancer efficacy. However, the correlations of molecular structure with charge-reversal property, tumor penetration, and drug delivery efficiency are unknown. Herein, aminopeptidase N (APN)-responsive conjugates were synthesized to investigate these correlations. We found that the monomeric unit structure and the polymer chain structure determined the enzymatic hydrolysis and charge-reversal rates, and accordingly, the transcytosis and tumor accumulation and penetration of the APN-responsive conjugates. The conjugate with moderate APN responsiveness balanced the in vitro transcytosis and in vivo overall drug delivery process and achieved the best tumor delivery efficiency, giving potent antitumor efficacy. This work provides new insight into the design of tumor enzyme-responsive charge-reversal nanomedicines for efficient cancer drug delivery.     

A DNA Nano-train Carrying a Predefined Drug Combination for Cancer Therapy

Herein, we reported a tumor cell-targeting aptamer-nano-train to deliver paclitaxel(PTX) and combretastatin A4(CA4) at a predefined ratio to cancer cells based on DNA nanotechnology. Such a drug-carrying aptamer-nano-train(aptamer-NT-PTX/CA4) was prepared via self-assembly of two DNA hairpins, which were conjugated with PTX and CA4, respectively, induced by aptamer trigger. Our research revealed that the aptamer-NT-PTX/CA4 could specifically recognize CD71-positive cancer cells, but not CD71-negative healthy normal cells, and achieve synergistic therapeutic effect on cancer cells. The aptamer-nano-train-based strategy is simple and efficient, and provides a new platform for drug combination cancer therapy.     

Self-Assembled Peptidyl Aggregates for the Fluorogenic Recognition of Mitochondrial DNA G-Quadruplexes

The selective monitoring of G-quadruplex (G4) structures in living cells is important to elucidate their functions and reveal their value as diagnostic or therapeutic targets. Here we report a fluorogenic probe ( CV2 ) able to selectively light-up parallel G4 DNA over antiparallel topologies. CV2 was constructed by conjugating the excimer-forming CV dye with a peptide sequence (l -Arg-l -Gly-glutaric acid) that specifically recognizes G4s. CV2 forms self-assembled, red excimer-emitting nanoaggregates in aqueous media, but specific binding to G4s triggers its disassembly into rigidified monomeric dyes, leading to a dramatic fluorescence enhancement. Moreover, selective permeation of CV2 stains G4s in mitochondria over the nucleus. CV2 was employed for tracking the folding and unfolding of G4s in living cells, and for monitoring mitochondrial DNA (mtDNA) damage. These properties make CV2 appealing to investigate the possible roles of mtDNA G4s in diseases that involve mitochondrial dysfunction.     

The Healing Capability of Clove Flower Extract (CFE) in Streptozotocin-Induced (STZ-Induced) Diabetic Rat Wounds Infected with Multidrug Resistant Bacteria

Treatment of diabetic foot ulcer (DFU) is of great challenge as it is shown to be infected by multidrug resistant bacteria (MDR bacteria). Sixty four bacterial isolates were isolated from DFU cases; antibiotic susceptibility tests were carried out for all of them. One bacterial isolate (number 11) was shown to resist the action of 8 out of 12 antibiotics used and was identified by both a Vitek-2 system and 16S rRNA fingerprints as belonging to Proteus mirabilis, and was designated Proteus mirabilis {"type":"entrez-nucleotide","attrs":{"text":"LC587231","term_id":"1917123988"}}LC587231 (P. mirabilis). Clove flower extract (CFE) inhibited distinctively the P. mirabilis bacterium obtained. GC-MS spectroscopy showed that this CFE contained nine bioactive compounds. The effect of CFE on wound healing of Type 1 diabetic albino rats (Rattus norvegicus) was studied. The results indicated that topical application of CFE hydrogel improved wound size, wound index, mRNA expression of the wound healing markers (Coli1, MMP9, Fibronectin, PCNA, and TGFβ), growth factor signaling pathways (PPAR-α, PGC1-α, GLP-1, GLPr-1, EGF-β, EGF-βr, VEGF-β, and FGF-β), inflammatory cytokine expression (IL8, TNFα, NFKβ, IL1β, and MCP1), as well as anti-inflammatory cytokines (IL4 & IL10), pro-apoptotic markers (FAS, FAS-L, BAX, BAX/BCL-2, Caspase-3, P53, P38), as well as an antiapoptotic one (BCL2). Furthermore, it improved the wound oxidative state and reduced the wound microbial load, as the cefepime therapy improved the wound healing parameters. Based on the previous notions, it could be concluded that CFE represents a valid antibiotics alternative for DFU therapy since it improves diabetic wound healing and exerts antibacterial activity either in vitro or in vivo.     

Efficient preparation of amine-modified oligodeoxynucleotide using modified H-phosphonate chemistry for DNA microarray fabrication

Amine-modified oligodeoxynucleotides (AMO) are commonly used probe oligodeoxynucleotides for DNA microarray preparation. Two methods are currently used for AMO preparation—use of amine phosphoramidites protected by acid-labile monomethoxytrityl (MMT) groups or alkali-labile trifluoroacetyl (TFA) groups. Because conventional AMO preparation procedures have defects, for example stringent acidic conditions are required for deprotection of MMT and hydrophobic purification cannot be used for TFA-protected amino groups, conventional preparation of AMO is unlikely to result in the expected outcome. In this paper a method of AMO synthesis using modified H-phosphonate chemistry is suggested. An aliphatic diamine is coupled with a phosphonate group forming a phosphoramidate linkage to the last internucleotide phosphate of oligodeoxynucleotides. In this method dimethoxytrityl (DMT) purification steps are used and stringent acid deprotection is not required to obtain the AMO. Although the method could lead to formation of AMO diastereomers, melting-temperature and CD analysis showed for two AMO that DNA duplex formation was the same as when normal oligodeoxynucleotides were used. Also, when these AMO were used as probes for DNA microarrays the immobilization efficiency was similar to that for AMO probes prepared by conventional means using an amino-modifier unit. The hybridization performance of these AMO was better than for those prepared conventionally. The procedures suggested would be useful for preparation of efficient AMO for fabrication of DNA microarrays and DNA-based nanoparticle systems. Nagendra Kumar Kamisetty and Seung Pil Pack have equally contributed to this work.     

A Metabolic-Tag-Based Method for Assessing the Permeation of Small Molecules Across the Mycomembrane in Live Mycobacteria**

The general lack of permeability of small molecules observed for Mycobacterium tuberculosis (Mtb) is most ascribed to its unique cell envelope. More specifically, the outer mycomembrane is hypothesized to be the principal determinant for access of antibiotics to their molecular targets. We describe a novel assay that combines metabolic tagging of the peptidoglycan, which sits directly beneath the mycomembrane, click chemistry of test molecules, and a fluorescent labeling chase step, to measure the permeation of small molecules. We showed that the assay workflow was robust and compatible with high-throughput analysis in mycobacteria by testing a small panel of azide-tagged molecules. The general trend is similar across the two types of mycobacteria with some notable exceptions. We anticipate that this assay platform will lay the foundation for medicinal chemistry efforts to understand and improve uptake of both existing drugs and newly-discovered compounds into mycobacteria.     

DNA origami nanostructures for controlled therapeutic drug delivery

DNA nanostructures are emerging as a versatile platform for controlled drug delivery as a result of recent progress in production yield and strategies to obtain prolonged stability in biological environments. The construction of nanostructures from this unique biomaterial provides unparalleled control over structural and functional parameters. Recent applications of DNA origami-based nanocarriers for therapeutic drug delivery in preclinical phases highlight them as promising alternatives to conventional nanomaterials, as they benefit from the inherent favorable properties of DNA including biocompatibility and precise spatial addressability. By incorporating targeting aptamers and responsive properties into the nanocarrier design, more selective DNA origami-based nanocarriers are successfully prepared. On the other hand, current systems remain poorly understood in terms of biodistribution, final fate, and controlled drug release. As such, advances are needed to translate this material platform in its full potential for therapeutic applications.     

Efficient and sensitive method of DNA silver staining in polyacrylamide gels

DNA silver staining is widely used to detect DNA fragment in polyacrylamide gel with high sensitivity. Conventional procedures of the silver staining involve several steps, which take about 40 min to 2 h in total. To improve the efficiency of DNA silver staining, a more efficient protocol is developed in this study. The procedure comprises only four steps including impregnating, rinsing, developing, and stopping, and could be completed within 20 min. Nitric acid and ethanol in the silver-impregnation step of the new procedure eliminates the need for prior treatment of gels with a fixing solution and following rinse prior to impregnation with silver. The procedure has high sensitivity and long storage lifetime. The minimum detectable mass of DNA is 0.44 and 3.5 ng in denaturing and nondenaturing polyacrylamide gel, respectively.     

A modified uridine for the synthesis of branched DNA

Branched DNA constructs have found wide application in DNA-based nanotechnology. Several reports describe the generation of branched DNA structures with variable numbers of arms to self-assemble with pre-designed architectures. Branched DNA is generated by using designed rigid crossover DNA molecules as building blocks. Alternatively, branched DNAs can also be generated by using synthetic branch points derived either from nucleoside or non-nucleoside building blocks. Herein, we report the synthesis of modified uridine derivatives as branching monomer for the synthesis of branched DNA and first studies of their self-assembling properties.     

A thermostable azo-linker for reversible photoregulation of DNA replication

Small molecules such as azobenzenes, one of the best reversible photo-switches, can be covalently incorporated into DNA to regulate its structures and functions with irradiation of the specific wavelengths. Using this strategy, a thermostable azobenzene linker was employed to construct modified oligodeoxynucleotides, and we successfully achieve reversible photoregulation of DNA replication in vitro with short irradiation time. Five minutes UV irradiation for regulating trans→cis transformation can minimize DNA damage, still ensure the polymerase reaction of cis-form. Formation of DNA hairpin structure can also be controlled by photoregulation using this linker.     

Efficient approach to produce multi‐functional copolymers for effective DNA binding

Norbornene‐derived copolymer with side‐chain phosphonic acid and cationic motifs (NORP‐PHOS‐CAT copolymer) are synthesized using ring opening metathesis polymerization method. All the monomers and polymers are characterized very carefully using nuclear magnetic resonance, MASS, Fourier transform infrared spectroscopy, and gel permeation chromatography technique. Thermogravimetric analysis, Fourier transform infrared spectroscopy, and scanning electron microscope techniques are employed to confirm the anchoring of Fe₃O₄ particles to the NORP‐PHOS‐CAT copolymer. Formation of nano‐aggregates from NORP‐PHOS‐CAT copolymer is first observed in dynamic light scattering, and later, it is confirmed as nano‐spheres by scanning electron microscope and transmission electron microscopy studies. Zeta‐potential values of nano‐spheres suggest that the cationic motifs are on the surface. The encapsulation of anionic dye methyl orange to the nanocarrier is analyzed through UV–Vis spectroscopy in aqueous medium. DNA binding nature of NORP‐PHOS‐CAT‐Fe copolymer is confirmed through the circular dichroism measurement and UV–Vis spectroscopy. To the best of our knowledge, this is the first report where the norbornene‐derived copolymers that are elegantly synthesized with both magnetic as well as cationic nature to demonstrate the effective encapsulation of dye into the nano‐spheres followed by its release. Copyright © 2016 John Wiley & Sons, Ltd.     

基于DNA纳米花的细胞自噬基因沉默用于增敏抗肿瘤化疗

自噬是真核细胞降解蛋白质的重要途径之一,在细胞的更新代谢中起重要作用.肿瘤细胞借助高水平的细胞自噬能够阻断细胞凋亡途径,降低化疗药物的抗肿瘤效果.本文通过设计编码有核酸适配体序列(Aptamer)和DNA酶序列(DNAzyme)的多功能DNA纳米花,利用DNA序列可负载化疗药物阿霉素(Dox)的特性,实现了对肿瘤细胞特异靶向的药物递送,并高效沉默肿瘤细胞的自噬相关基因ATG5,达到增敏抗肿瘤化疗的效果.通过RT-PCR实验验证合成的DNA纳米花可以有效剪切肿瘤细胞中自噬相关基因ATG5的mRNA;并通过DNA纳米花的细胞毒性和细胞凋亡实验研究了其对肿瘤细胞系MCF-7的靶向治疗作用,结果显示该多功能DNA纳米花在增敏抗肿瘤化疗方面具有明显优势.     

A general approach for rational design of fluorescent DNA aptazyme sensors based on target-induced unfolding of DNA hairpins

DNA aptazymes are allosteric DNAzymes activated by the targets of DNA aptamers. They take the advantages of both aptamers and DNAzymes, which can recognize specific targets with high selectivity and catalyze multiple-turnover reactions for signal amplification, respectively, and have shown their great promise in many analytical applications. So far, however, the available examples of DNA aptazyme sensors are still limited in utilizing only several DNAzymes and DNA aptamers, most likely due to the lack of a general and simple approach for rational design. Herein, we have developed such a general approach for designing fluorescent DNA aptazyme sensors. In this approach, aptamers and DNAzymes are connected at the ends to avoid any change in their original sequences, therefore enabling the general use of different aptamers and DNAzymes in the design. Upon activation of the aptazymes by the targets of interest, the rate of fluorescence enhancement via the cleavage of a dually labeled substrate by the active aptazymes is then monitored for target quantification. Two DNAzymes and two aptamers are used as examples for the design of three fluorescent aptazyme sensors, and they all show high selectivity and sensitivity for the detection of their targets. More DNA aptazyme sensors for a broader range of targets could be developed by this general approach as long as suitable DNAzymes and aptamers are used.