Multi-Hydrogen Bonding on Quaternized-Oligourea Receptor Facilitated Its Interaction with Bacterial Cell Membranes and DNA for Broad-Spectrum Bacteria Killing.

Herein, we report a new strategy for the design of antibiotic agents based on the electrostatic interaction and hydrogen bonding, highlighting the significance of hydrogen bonding and the increased recognition sites in facilitating the interaction with bacterial cell membranes and DNA. A series of quaternary ammonium functionalized urea-based anion receptors were studied. While the monodentate mono-urea M1, bisurea M2, and trisurea M3 failed to break through the cell membrane barrier and thus could not kill bacteria, the extended bidentate dimers D1-D3 presented gradually increased membrane penetrating capabilities, DNA conformation perturbation abilities, and broad-spectrum antibacterial activities against E. coli, P. aeruginosa, S. aureus, E. faecalis, and S. epidermidis.

A non-enzymatic, isothermal amplification sensor for quantifying the relative abundance of Akkermansia muciniphila.

Herein, we have developed a non-enzymatic, isothermal amplification assay (NIA sensor) based on a catalytic hairpin assembly (CHA) reaction for quantifying the relative abundance of Akkermansia muciniphila. Through detection of the MUC-1437 gene (limit of detection: 8.3 fM) in a dynamic range from 10 fM to 1 nM, the NIA sensor shows high sensitivity and selectivity in preclinical models of mice fed a normal or high-fat diet (HFD), and treated with antibiotics (ATB). The NIA sensor, which operates without the use of any enzymes, leading to simplicity and cost-effectiveness, has great potential for biosensing research and clinical diagnostic applications.

DNA Framework-Templated Fabrication of Ultrathin Electroactive Gold Nanosheets.

Generally, two-dimensional gold nanomaterials have unique properties and functions that offer exciting application prospects. However, the crystal phases of these materials tend to be limited to the thermodynamically stable crystal structure. Herein, we report a DNA framework-templated approach for the ambient aqueous synthesis of freestanding and microscale amorphous gold nanosheets with ultrathin sub-nanometer thickness. We observe that extended single-stranded DNA on DNA nanosheets can induce site-specific metallization and enable precise modification of the metalized nanostructures at predefined positions. More importantly, the as-prepared gold nanosheets can serve as an electrocatalyst for glucose oxidase-catalyzed aerobic oxidation, exhibiting enhanced electrocatalytic activity (~3-fold) relative to discrete gold nanoclusters owing to a larger electrochemical active area and wider band gap. The proposed DNA framework-templated metallization strategy is expected to be applicable in a broad range of fields, from catalysis to new energy materials.

Self-assembly of Copper Nanoclusters Using DNA Nanoribbon Templates for Sensitive Electrochemical Detection of H2O2 in Live Cells.

The excessive secretion of H2O2 within cells is closely associated with cellular dysfunction. Therefore, high sensitivity in situ detection of H2O2 released from living cells was valuable in clinical diagnosis. In the present work, a novel electrochemical cells sensing platform by synthesizing copper nanoclusters (CuNCs) at room temperature based on DNA nanoribbon (DNR) as a template (DNR-CuNCs). The tight and ordered arrangement of nanostructured assemblies of DNR-CuNCs conferred the sensor with superior stability (45 days) and electrochemical performance. The MUC1 aptamer extending from the DNR template enabled the direct capture MCF-7 cells on electrode surface, this facilitated real-time monitoring of H2O2 release from stimulated MCF-7 cells. While the captured MCF-7 cells on the electrode surface significantly amplified the current signal of H2O2 release compared with the traditional electrochemical detection H2O2 released signal by MCF-7 cells in PBS solution. The approach provides an effective strategy for the design of versatile sensors and achieving monitored cell release of H2O2 in long time horizon (10 h). Thereby expanding the possibilities for detecting biomolecules from live cells in clinical diagnosis and biomedical applications.

Copper Nanosheet-Based Wash-Free Fluorescence Imaging of Cancer Cells.

Near-infrared fluorescence (NIRF) probes greatly facilitate in vivo imaging of various biologically important species. However, there are several significant limitations such as consuming washing steps, photobleaching, and low signal intensity. Herein, we synthesized fluorescent copper nanosheets templated with DNA scaffolds (DNS/CuNSs). We employ them and Cy5.5 of the fluorescence resonance energy transfer (FRET) system, which have a larger Stokes shift (∼12-fold) than the traditional NIRF dye Cy5.5. Based on their excellent fluorescence properties, we employ DNS/CuNSs-Cy5.5 for fluorescence probes in cancer cell imaging. Compared with the free Cy5.5 fluorescence probe, the novel fluorescence imaging probe implements wash-free imaging and exhibits enhanced anti-photobleaching ability (∼5.5-fold). Moreover, the FRET system constructed by DNS/CuNSs has a higher signal amplification ability (∼4.17-fold), which is more similar to that of Cu nanoclusters prepared with DNA nanomonomers as a template. This work provides a new idea for cancer cell MCF-7 imaging and is expected to promote the development of cancer cell fluorescence imaging.

DNA Nanoribbon-Assisted Intracellular Biosynthesis of Fluorescent Gold Nanoclusters for Cancer Cell Imaging.

Metal nanoclusters (NCs) have emerged as a promising class of fluorescent probes for cellular imaging due to their high resistance to photobleaching and low toxicity. Nevertheless, their widespread use in clinical diagnosis is limited by their unstable intracellular fluorescence. In this study, we develop an intracellularly biosynthesized fluorescent probe, DNA nanoribbon-gold NCs (DNR/AuNCs), for long-term cellular tracking. Our results show that DNR/AuNCs exhibit a 4-fold enhancement of intracellular fluorescence intensity compared to free AuNCs. We also investigated the mechanism underlying the fluorescence enhancement of AuNCs by DNRs. Our findings suggest that the higher synthesis efficiency and stability of AuNCs in the lysosome may contribute to their fluorescence enhancement, which enables long-term (up to 15 days) fluorescence imaging of cancer cells (enhancement of ∼60 times compared to free AuNCs). Furthermore, we observe similar results with other metal NCs, confirming the generality of the DNR-assisted biosynthesis approach for preparing highly bright and stable fluorescent metal NCs for cancer cell imaging.

Self-assembly Induced Enhanced Electrochemiluminescence of Copper Nanoclusters Using DNA Nanoribbon Templates.

Copper nanoclusters (CuNCs) are attractive electrochemiluminescence (ECL) emitters as Cu is comparatively inexpensive, nontoxic, and highly abundant. However, their ECL yield is relatively low. Herein, we report that orderly self-assembly of CuNCs using DNA nanoribbon as the template (DNR/CuNCs) conferred the CuNCs with improved ECL properties compared with individual CuNCs in both annihilation and co-reactant processes. The DNR/CuNCs resulted in a high ECL yield of 46.8 % in K2 S2 O8 , which was ≈68 times higher than that of individual CuNCs. This strategy was successfully extended to other ECL emitters, such as gold nanoclusters and the Ru(bpy)3 2+ /TPrA system. Furthermore, as an application of DNR/CuNCs, a DNR/CuNC-based ECL biosensor with higher sensitivity was constructed for dopamine determination (two orders of magnitude lower than that previously reported), showing that DNR/CuNCs have a potential for application in ECL bioanalysis as a new type of superior luminophore candidate.

Micron-Scale Fabrication of Ultrathin Amorphous Copper Nanosheets Templated by DNA Scaffolds.

Two-dimensional (2D) amorphous materials could outperform their crystalline counterparts toward various applications because they have more defects and reactive sites and thus could exhibit a unique surface chemical state and provide an advanced electron/ion transport path. Nevertheless, it is challenging to fabricate ultrathin and large-sized 2D amorphous metallic nanomaterials in a mild and controllable manner due to the strong metallic bonds between metal atoms. Here, we reported a simple yet fast (10 min) DNA nanosheet (DNS)-templated method to synthesize micron-scale amorphous copper nanosheets (CuNSs) with a thickness of 1.9 ± 0.4 nm in aqueous solution at room temperature. We demonstrated the amorphous feature of the DNS/CuNSs by transmission electron microscopy (TEM) and X-ray diffraction (XRD). Interestingly, we found that they could transform to crystalline forms under continuous electron beam irradiation. Of note, the amorphous DNS/CuNSs exhibited much stronger photoemission (∼62-fold) and photostability than dsDNA-templated discrete Cu nanoclusters due to the elevation of both the conduction band (CB) and valence band (VB). Such ultrathin amorphous DNS/CuNSs hold great potential for practical applications in biosensing, nanodevices, and photodevices.

Snowflake-like DNA crystals templated Cu clusters as a fluorescent turn-on probe for sensing actin.

Herein, we synthesized snowflake-like DNA crystals (SDC) via hybridization chain reaction and used it for the first time in the synthesis of copper nanoclusters with enhanced fluorescence. Atomic force microscopy (AFM) and laser confocal microscopy characterization confirmed that SDC/CuNCs are self-assembled successfully on SDC. Aggregation induced emission allows SDC/CuNCs to exhibit better stability and stronger emission intensity. Thus, we developed the "turn-on" label-free fluorescence detection method of actin based on SDC/CuNCs which offer simplicity, low cost, good selectivity, and high sensitivity. The detection limit was determined to be 0.0124 µg mL-1, which was an order of magnitude lower than that of reported fluorescent methods (0.12 µg mL-1). Compared with previous method, the linear range is also much wider. We also performed standard recovery experiments in actual samples for evaluating the practicality of this strategy and proved that the capability of the proposed approach for the determination of actin is feasible and the interference from complex biological samples is negligible. These results indicate that SDC/CuNCs are expected to play a more important role in the field of biosensors.

DNA Nanoribbon-Templated Self-Assembly of Ultrasmall Fluorescent Copper Nanoclusters with Enhanced Luminescence.

Fluorescent copper nanoclusters (CuNCs) have been widely used in chemical sensors, biological imaging, and light-emitting devices. However, individual fluorescent CuNCs have limitations in their capabilities arising from poor photostability and weak emission intensities. As one kind of aggregation-induced emission luminogen (AIEgen), the formation of aggregates with high compactness and good order can efficiently improve the emission intensity, stability, and tunability of CuNCs. Here, DNA nanoribbons, containing multiple specific binding sites, serve as a template for in situ synthesis and assembly of ultrasmall CuNCs (0.6 nm). These CuNC self-assemblies exhibit enhanced luminescence and excellent fluorescence stability because of tight and ordered arrangement through DNA nanoribbons templating. Furthermore, the stable and bright CuNC assemblies are demonstrated in the high-sensitivity detection and intracellular fluorescence imaging of biothiols.

Functional modulation of cytochrome C upon specific binding to DNA nanoribbons.

We discovered that the function of cytochrome C can be modulated by DNA nanoribbons. Meanwhile, the interplay between the DNA nanoribbons and the native cytochrome C and the possible mechanisms are also discussed.

Solving mazes with single-molecule DNA navigators.

Molecular devices with information-processing capabilities hold great promise for developing intelligent nanorobotics. Here we demonstrate a DNA navigator system that can perform single-molecule parallel depth-first search on a ten-vertex rooted tree defined on a two-dimensional DNA origami platform. Pathfinding by the DNA navigators exploits a localized strand exchange cascade, which is initiated at a unique trigger site on the origami with subsequent automatic progression along paths defined by DNA hairpins containing a universal traversal sequence. Each single-molecule navigator autonomously explores one of the possible paths through the tree. A specific solution path connecting a given pair of start and end vertices can then be easily extracted from the set of all paths taken by the navigators collectively. The solution path laid out on origami is illustrated with single-molecule imaging. Our approach points towards the realization of molecular materials with embedded computational functions operating at the single-molecule level.

Docking of Antibodies into the Cavities of DNA Origami Structures.

Immobilized antibodies are extensively employed for medical diagnostics, such as in enzyme-linked immunosorbent assays. Despite their widespread use, the ability to control the orientation of immobilized antibodies on surfaces is very limited. Herein, we report a method for the covalent and orientation-selective immobilization of antibodies in designed cavities in 2D and 3D DNA origami structures. Two tris(NTA)-modified strands are inserted into the cavity to form NTA-metal complexes with histidine clusters on the Fc domain. Subsequent covalent linkage to the antibody was achieved by coupling to lysine residues. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) confirmed the efficient immobilization of the antibodies in the origami structures. This increased control over the orientation of antibodies in nanostructures and on surfaces has the potential to direct the interactions between antibodies and targets and to provide more regular surface assemblies of antibodies.

A DNA nanoribbon as a potent inhibitor of metallo-ß-lactamases.

We discovered a promising metallo-ß-lactamase inhibitor, a DNA nanoribbon, by enzymatic kinetics and isothermal titration calorimetry evaluations. Atomic force microscopy, gel electrophoresis, competitive binding experiments, circular dichroic and thermal denaturation studies suggested that the DNA nanoribbon could bind to the enzyme through a minor groove.

DNA Nanostructure as Smart Carriers for Drug Delivery.

Self-assembled DNA nanostructures have recently emerged as a type of drug delivery carriers due to their suitable sizes, well-defined nanoscale shapes, precise spatial addressability, and excellent biocompatibility. Here, we describe practical procedures in detail for the design and construction of DNA nanostructures with different width and patterns by long rolling circle amplification (RCA) strands and a few short staples, and provide practical guidance and troubleshooting advice for delivering CpG immunostimulatory drugs with these RCA based DNA nanostructures.

Growth and origami folding of DNA on nanoparticles for high-efficiency molecular transport in cellular imaging and drug delivery.

A novel three-dimensional (3D) superstructure based on the growth and origami folding of DNA on gold nanoparticles (AuNPs) was developed. The 3D superstructure contains a nanoparticle core and dozens of two-dimensional DNA belts folded from long single-stranded DNAs grown in situ on the nanoparticle by rolling circle amplification (RCA). We designed two mechanisms to achieve the loading of molecules onto the 3D superstructures. In one mechanism, ligands bound to target molecules are merged into the growing DNA during the RCA process (merging mechanism). In the other mechanism, target molecules are intercalated into the double-stranded DNAs produced by origami folding (intercalating mechanism). We demonstrated that the as-fabricated 3D superstructures have a high molecule-loading capacity and that they enable the high-efficiency transport of signal reporters and drugs for cellular imaging and drug delivery, respectively.

Self-assembly of DNA-based drug delivery nanocarriers with rolling circle amplification.

DNA nanostructures have recently emerged as a type of drug delivery nanocarriers due to their suitable sizes, well-defined structures and low-toxicity. Here, we present a protocol for the assembly of DNA nanoribbon structures with rolling circle amplification (RCA) and delivery of CpG oligonucleotide. DNA nanoribbons with different dimensions and patterns were assembled with long RCA strands and several short staples. Significantly, we demonstrated they exhibited high-efficiency cellular uptake and improved immunostimulatory activity compared with ss- or ds- DNA.

Exploiting the higher specificity of silver amalgamation: selective detection of mercury(II) by forming Ag/Hg amalgam.

Heavy metal ion pollution poses severe risks in human health and the environment. Driven by the need to detect trace amounts of mercury, this article demonstrates, for the first time, that silver/mercury amalgamation, combining with DNA-protected silver nanoparticles (AgNPs), can be used for rapid, easy and reliable screening of Hg(2+) ions with high sensitivity and selectivity over competing analytes. In our proposed approach, Hg(2+) detection is achieved by reducing the mercury species to elemental mercury, silver atoms were chosen as the mercury atoms' acceptors by forming Ag/Hg amalgam. To signal fluorescently this silver amalgamation event, a FAM-labeled ssDNA was employed as the signal reporter. AgNPs were grown on the DNA strand that resulted in greatly quenching the FAM fluorescence. Formation of Ag/Hg amalgam suppresses AgNPs growth on the DNA, leading to fluorescence signal increase relative to the fluorescence without Hg(2+) ions, as well as marked by fluorescence quenching. This FAM fluorescence enhancement can be used for detection of Hg(2+) at the a few nanomolar level. Moreover, due to excellent specificity of silver amalgamation with mercury, the sensing system is highly selective for Hg(2+) and does not respond to other metal ions with up to millimolar concentration levels. This sensor is successfully applied to determination of Hg(2+) in tap water, spring water and river water samples. The results shown herein have important implications in the development of new fluorescent sensors for the fast, easy, and selective detection and quantification of Hg(2+) in environmental and biological samples.

Rolling circle amplification-based DNA origami nanostructrures for intracellular delivery of immunostimulatory drugs.

Several single-stranded scaffold DNA, obtained from rolling circle amplification (RCA), are folded by different staples to form DNA nanoribbons. These DNA nanoribbons are rigid, simple to design, and cost-effective drug carriers, which are readily internalized by mammalian cells and show enhanced immunostimulatory activity.

A carbon nanoparticle-based low-background biosensing platform for sensitive and label-free fluorescent assay of DNA methylation.

Combining a DNA intercalator, SYBR Green I, and enzyme-linkage reactions with carbon nanoparticles, a low-background biosensing platform for label-free and sensitive fluorescent assay of DNA methylation is reported.