Dendritic DNA and its application to biological and biomedical technology

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Dendrimer has been attractive as fundamental materials for applications in the research fields such as nanomaterial, biomedicine, tissue engineering and so on, due to the highly defined chemical structure and composition. Though the organic and inorganic dendrimers achieved complex functionalization, the synthetic difficulty and biocompatibility remained a great challenge for further application in medicine and biotechnology. DNA possesses a desirable biocompatibility as a polymeric material and an unique programmable property. By taking the advantages of these features, attempts to develop a dendritic DNA self-assembled dendrimer platform have led my research work for the application of nanoscale self-assembly, DNA nanotechnology, drug delivery, gene therapy and bio-scaffold, which were elaborated in the following chapters, respectively. Attempts to apply long-stretch of G-quadruplex for electric nanowire have spurred the questions of the distance dependence of charge transport (CT) efficiency. In Chapter 2, a series of tetra-stranded G-quadruplex assembled with a dendritic DNA architecture to explore efficient charge transport was observed via tetra-stranded G-quadruplex over 28Å. Charge transport was more accelerated and achieved higher efficiency over longer G-quadruplex bridges than that of duplex DNA with the most CT optimal sequence, adenine tract. Higher electron transport efficiency suggests tetra-stranded G-quadruplexes hold promising potentials as molecular electric nanowire in DNA-based electric nanowire and nanodevice. Hydrogels constructed by nucleic acids possess great potentials in biological and biomedical research. In Chapter 3, we developed a DNA hydrogel via self-assembly of a dendritic DNA architecture. Due to the 3D structure of dendritic DNA, the hydrogel showed tunable mechanical stiffness and robust reversible thixotropy even under a nanomolar DNA concentration. Rapid isothermal assembly of the dendritic DNA allowed the gelation of culture medium at an ambient condition. Both cancerous and somatic cells proliferated in DNA hydrogel with higher or equal efficacy to those in tranditional 2D solution culture. Complex cell morphologies such as cell spheroids can be manipulated in gel state of cell medium. Bi-entity of dendritic branches enabled the specific loading of bioactive clues like aptamers to the DNA hydrogel with intact biological activity, and regulate cell growth. Thus, dendritic DNA assembled hydrogel can be an ideal biomaterial as a robust, readily functionalizable and easy-casting biomaterials for 3D cell culture in the fields of tissue engineering, biomedicine, etc. Inspired the excellent effect of the synergetic therapy, a universal platform for the simultaneous delivery of small molecular chemodrugs, gene therapy and bioimaging for synchronous diagnosis and therapy was developed from a self-assembly DNA nanohydrogel. The detail characterization of the DNA nanohydrogel was illustrated in chapter 4. The factors that could affect the nucleation, composition and morphology of DNA nanohydrogel, such as gelation concentration, component ratio and sequence were explored. DNA nanohydrogel has a good long-term stability against aggregation in aqueous buffer solution and over repeatable dehydration/swell, and high thermostability for the biomedical/biotechnological protocol at physiological temperature. DNA nanohydrogel showed the high selectivity of cellular uptaken towards cancerous cells with little internalization to somatic cells. The low cell cytotoxicity to both malignant and healthy cell lines indicated the DNA nanohydrogel was a promising nontoxic nanoscale vessel for drug delivery. Meanwhile, 2 hr life time in blood plasma showed that DNA nanohydrogel held good biodegradability and would have decent stability for in vivo applications. In chapter 5, the DNA nanohydrogel was explored for three functions for drug delivery, gene therapy and diagnostic bioimaging. Small chemotherapy drug, doxorubicin, could be loaded into the DNA nanohydrogel with a much higher efficiency than the current existing DNA-base nanoplatform for drug delivery. Slow release of doxorubicin from DNA nanohydrogel was observed over the time span of hours. With aptamer decorated via loading branch of dendritic DNA, DNA nanohydrogel showed specific targeting to cancerous cell lines. The dual-modified DNA nanohydrogel with aptamer and doxorubicin exhibited great selectivity and cytotoxicity on specific cancerous cell lines, while the cytotoxicity of doxorubicin towards healthy cell lines were significantly minimized Alternatively, the DNA nanohydrogel can be applied for gene therapy by coding an antisense oligonucleotide sequence on the loading arm of dendric DNA to down regulate the mRNA expression of survivin gene, which is overexpressed in almost all the cancerous cells. The down regulation of surviving gene expression by DNA nanohydrogel was efficient and the cytotoxicity was specific to the cancerous cells. By labelled with fluorescent dye, the DNA nanohydrogel was used to image the subcellular level and discribution of survivin mRNA. In conclusion, a dendritic DNA as building block is developed and applied for biological and biomedical research. The unique spiral, flexible, three-dimensional structure of the building block enables a series of biocompatible materials and endows a vast potential in drug delivery, gene therapy, bioimaging, nanostructure assembly, etc.