Development of Multifunctional DNA-based Nanomaterials for Biomedical and Nanotechnological Applications

多功能的DNA納米材料開發及其在生物醫學和納米技術的應用

Student thesis: Doctoral Thesis

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Award date21 Dec 2020

Abstract

DNA is a biological polymer that stores and transmits genetic information in biological systems. The field of DNA nanotechnology takes DNA out of its biological context, utilizes its information to assemble structural units and joins them together. This field has a significant impact on nanoscience and nanotechnology, and has advanced to allow control of molecular self-assemblies. Time and resources have been channeled to investigate logic gate operations, drug carriers and cargo transporters. New DNA devices are kept on advancing based on previous art pieces of different scientists. Herein, I have demonstrated (1) a DNA drug carrier that can slow down the growth of brain tumors in vivo, (2) a DNA nanostructure that involves a G-quadruplex as an operation mechanism for cholesterol detection, (3) a recyclable 2D triangular rung logic OR gate operated by ultra-violet (UV) and near infra-red (NIR) wavelengths, and (4) a simple double stranded logic gate based on G-quadruplex and light-sensitive moieties.

In this thesis, I used DNA nanocages as drug carriers for glioma tumour treatment and for detection of cholesterol in blood. We have shown that DNA nanocages can pass through blood brain barrier (BBB) layer in an in vitro transwell test; moreover, we show that after intravascular injection our DNA nanocage can pass through the BBB in vivo without the assistance of targeting aptamers. We also compared the permeabilities of DNA nanocages and DNA nanotubes to investigate the effects of the size of DNA nanostructures on BBB transversion. Our results showed that smaller sized nanocages passed through the in vitro BBB models better. Intravascular injection of Doxorubicin loaded cage successfully reduced the glioma tumour size by which makes DNA nanocarriers an exciting candidate for drug carriers in the treatment of brain diseases.

In Chapter 3, DNA nanocages were used as switches by integrating a G-quadruplex sequence. The cage can be switched on and off by cations and a cation chelating agent (2,2,2 cryptand). After adding potassium ions and hemins, the DNA nanocage further became a DNAzyme for catalyzing the decomposition of hydrogen peroxide. With the aid of cholesterol oxidase and 2,2'-azino-bis(3-ethylbenzothiazoline6-sulfonic acid) (ABTS) reagent, DNA nanocages can be used for colorimetric detection of cholesterol. The DNA switches had greater nuclease resistance than the single stranded G- rich sequence making the G-quadruplex containing DNA nanostructures more stable than the single stranded G-quadruplex in a biological environment.

Chapters 4 and 5 describe the development of photoactivated logic gates. The first generation of DNA logic gate was a 2D DNA triangular ring with 3 complementary strands. This setup employed the photocleavage molecule photocleavage (PC) spacer and 4-nitro-4’-phenoxy-1,1’-biphenyl (4-NB) and formed a logic OR gate. The OR gate can be operated by ultra-violet light or/and near infrared light and demonstrated with fluorescent and quenchers pair.

The second generation of our logic gates employed another two-photon cleavage molecule (2,7-bis-9,9-bis-[1-(3,6-dioxaheptyl)]-fluorene, BNSF) in which the one-photon maximum wavelength is red shifted compared with 4-NB, and has a higher uncaging cross section. With the integration of the BNSF and PC spacers, we further developed a series of photoactivated DNA logic gates by employing the DNA strand displacement and G-quadruplex: YES, NOT, NOR, OR, AND gates. The characterizations of the logic gates were performed with one-photon irradiation and need further investigation using two-photon irradiation as the input.