Hisakage Funabashi

A mechanical metamaterial made from a DNA hydrogel

Metamaterials are artificial substances that are structurally engineered to have properties not typically found in nature. To date, almost all metamaterials have been made from inorganic materials such as silicon and copper, which have unusual electromagnetic or acoustic properties that allow them to be used, for example, as invisible cloaks, superlenses or super absorbers for sound. Here, we show that metamaterials with unusual mechanical properties can be prepared using DNA as a building block. We used a polymerase enzyme to elongate DNA chains and weave them non-covalently into a hydrogel. The resulting material, which we term a meta-hydrogel, has liquid-like properties when taken out of water and solid-like properties when in water. Moreover, upon the addition of water, and after complete deformation, the hydrogel can be made to return to its original shape. The meta-hydrogel has a hierarchical internal structure and, as an example of its potential applications, we use it to create an electric circuit that uses water as a switch.

Multifunctional nanoarchitectures from DNA-based ABC monomers

The ability to attach different functional moieties to a molecular building block1,2 could lead to applications in nanoelectronics3, nanophotonics4, intelligent sensing5 and drug delivery6,7. The building unit needs to be both multivalent and anisotropic, and although many anisotropic building blocks have been created1,8,9,10,11,12, these have not been universally applicable. Recently, DNA has been used to generate various nanostructures13,14,15,16,17 or hybrid systems18,19,20,21,22,23,24,25, and as a generic building block for various applications26,27,28,29,30. Here, we report the creation of anisotropic, branched and crosslinkable building blocks (ABC monomers) from which multifunctional nanoarchitectures have been assembled. In particular, we demonstrate a target-driven polymerization process in which polymers are generated only in the presence of a specific DNA molecule, leading to highly sensitive pathogen detection. Using this monomer system, we have also designed a biocompatible nanovector that delivers both drugs and tracers simultaneously. Our approach provides a general yet versatile route towards the creation of a range of multifunctional nanoarchitectures. Supplementary information The online version of this article (doi:10.1038/nnano.2009.93) contains supplementary material, which is available to authorized users.

Biodegradable CpG DNA hydrogels for sustained delivery of doxorubicin and immunostimulatory signals in tumor-bearing mice.

Immunostimulatory CpG DNA was self-assembled to form DNA hydrogels for use as a sustained delivery system for both intercalated doxorubicin (DXR) and immunostimulatory CpG motifs for cancer treatment. X-shaped DNA (X-DNA) was designed as a building unit, and underwent ligation to form DNA hydrogels. Two types of X-DNA were constructed using four oligodeoxynucleotides each, one containing six potent CpG motifs (CpG X-DNA) and the other with none (CpG-free X-DNA). CpG X-DNA was more effective than its components or the CpG-free counterpart in terms of the production of tumor necrosis factor-α from murine macrophage-like RAW264.7 cells, as well as maturation of the murine dendritic DC2.4 cells. The cytotoxic effects of X-DNA, DXR and their complexes were examined in a co-culture system of colon26/Luc cells, a murine adenocarcinoma clone stably expressing firefly luciferase, and RAW264.7 cells. DXR/CpG X-DNA showed the highest ability to inhibit the proliferation of colon26/Luc cells. DXR was slowly released from CpG DNA hydrogels. Injections of DXR/CpG DNA hydrogels into a subcutaneous colon26 tumor effectively inhibited tumor growth. These results show that CpG DNA hydrogels are an effective sustained system for delivery of immunostimulatory signals to TLR9-positive immune cells and DXR to cancer cells.

A novel glucosylation reaction on anthocyanins catalyzed by acyl-glucose-dependent glucosyltransferase in the petals of carnation and delphinium.

This work describes a glucosylation reaction at the 5/7 positions of anthocyanins in the petals of carnations and delphiniums. Unusually, this reaction is catalyzed by acyl-glucose–dependent glucosyltransferases that belong to glycoside hydrolase family 1. This modification mechanism may play an important role in generating variation in anthocyanins. Glucosylation of anthocyanin in carnations (Dianthus caryophyllus) and delphiniums (Delphinium grandiflorum) involves novel sugar donors, aromatic acyl-glucoses, in a reaction catalyzed by the enzymes acyl-glucose–dependent anthocyanin 5(7)-O-glucosyltransferase (AA5GT and AA7GT). The AA5GT enzyme was purified from carnation petals, and cDNAs encoding carnation Dc AA5GT and the delphinium homolog Dg AA7GT were isolated. Recombinant Dc AA5GT and Dg AA7GT proteins showed AA5GT and AA7GT activities in vitro. Although expression of Dc AA5GT in developing carnation petals was highest at early stages, AA5GT activity and anthocyanin accumulation continued to increase during later stages. Neither Dc AA5GT expression nor AA5GT activity was observed in the petals of mutant carnations; these petals accumulated anthocyanin lacking the glucosyl moiety at the 5 position. Transient expression of Dc AA5GT in petal cells of mutant carnations is expected to result in the transfer of a glucose moiety to the 5 position of anthocyanin. The amino acid sequences of Dc AA5GT and Dg AA7GT showed high similarity to glycoside hydrolase family 1 proteins, which typically act as β-glycosidases. A phylogenetic analysis of the amino acid sequences suggested that other plant species are likely to have similar acyl-glucose–dependent glucosyltransferases.

The assembly of a short linear natural cytosine-phosphate-guanine DNA into dendritic structures and its effect on immunostimulatory activity.

DNA containing unmethylated CpG dinucleotides, or CpG motifs, (CpG DNA) has been explored as a therapeutic agent, owing to its potent immunostimulatory activity. A previous study showing that Y-shaped (Y-) CpG DNA has a high immunostimulatory activity compared with single- or double stranded CpG DNA suggests the possibility that CpG DNA in a more complicated structure is a stronger activator of the immune system. In the present study, dendrimer-like DNA (DL-DNA) was prepared by ligating Y-DNA monomers. The DL-DNA of the second or third generation with 12 or 24 highly potent CpG motifs in one unit, respectively, were designed and successfully prepared for the first time. These DL-DNAs induced greater amounts of tumor necrosis factor-alpha and interleukin-6 from RAW264.7 macrophage-like cells than did a mixture of Y-DNA with the same sequences as the corresponding DL-DNA. DL-DNA was more efficiently taken up by RAW264.7 cells than Y-DNA, but the increase was lower than that exhibited by the levels of cytokine release. These results suggest that the dendritic structure formation is a potential approach to increasing the immunostimulatory activity of CpG DNA without any modifications of the chemical structure of the natural phosphodiester DNA.

A Universal DNA-Based Protein Detection System

Protein immune detection requires secondary antibodies which must be carefully selected in order to avoid interspecies cross-reactivity, and is therefore restricted by the limited availability of primary/secondary antibody pairs. Here we present a versatile DNA-based protein detection system using a universal adapter to interface between IgG antibodies and DNA-modified reporter molecules. As a demonstration of this capability, we successfully used DNA nano-barcodes, quantum dots, and horseradish peroxidase enzyme to detect multiple proteins using our DNA-based labeling system. Our system not only eliminates secondary antibodies but also serves as a novel method platform for protein detection with modularity, high capacity, and multiplexed capability.

Non-destructive monitoring of rpoS promoter activity as stress marker for evaluating cellular physiological status

To monitor the extent of cellular physiological stress, the activity of the rpoS promoter was evaluated as a marker of the stress pathway. A reporter plasmid was constructed by inserting the GFPuv gene under the rpoS promoter and used to transform Escherichia coli cells. The fluorescence of the GFPuv protein was measured in intact cells in a non-destructive manner. The physiological status of the cells could be conveniently monitored using the rpoS-GFPuv reporter gene with respect to the cellular growth phase and to elevated ethanol and NaCl concentrations as two examples of environmental stress factors. Comparison of the response of different E. coli strains demonstrated an essential role of the relA gene in the induction of the rpoS-GFPuv reporter gene.

Noise-free accurate count of microbial colonies by time-lapse shadow image analysis

Microbial colonies in food matrices could be counted accurately by a novel noise-free method based on time-lapse shadow image analysis. An agar plate containing many clusters of microbial colonies and/or meat fragments was trans-illuminated to project their 2-dimensional (2D) shadow images on a color CCD camera. The 2D shadow images of every cluster distributed within a 3-mm thick agar layer were captured in focus simultaneously by means of a multiple focusing system, and were then converted to 3-dimensional (3D) shadow images. By time-lapse analysis of the 3D shadow images, it was determined whether each cluster comprised single or multiple colonies or a meat fragment. The analytical precision was high enough to be able to distinguish a microbial colony from a meat fragment, to recognize an oval image as two colonies contacting each other, and to detect microbial colonies hidden under a food fragment. The detection of hidden colonies is its outstanding performance in comparison with other systems. The present system attained accuracy for counting fewer than 5 colonies and is therefore of practical importance.

Construction of streptavidin-luciferase fusion protein for ATP sensing with fixed form

A fusion protein consisting of streptavidin and firefly luciferase was constructed to establish an accurate measuring technique of local ATP concentration. The fusion protein retained the binding ability of streptavidin and enzymatic activity of luciferase. Also, it could detect the concentration of antigens and could determine nanomolar concentrations of ATP in its fixed form via interactions with biotin-conjugated antibodies.

Porphyrin-uptake in liposomes and living cells using an exchange method with cyclodextrin

The water-solubilisation of porphyrin derivatives is very important for biological applications. Although liposomal drug carriers for porphyrin derivatives have shown significant promise in the field of medicinal chemistry (e.g., as sensitisers for photodynamic therapy), it is currently not possible to prepare lipid-membrane-incorporated tetraphenylporphyrin (TPP) with a high concentration of TPP using conventional methods. In this study, we have succeeded in preparing lipid-membrane-incorporated TPP and zinc(II) tetraphenylporphyrin (ZnTPP) from the corresponding TPP or ZnTPP·cyclodextrin complex using the exchange method in lipid-membranes composed of liposomes. Furthermore, the exchange method allowed for the incorporation of TPP or ZnTPP into the plasma membranes of HeLa cells. However, it was not possible to prepare lipid-membrane-incorporated porphyrin derivatives with polar and hydrophilic groups in the meso positions using this exchange reaction.