Aptamer-functionalized DNA nanostructures and applications in targeted cell delivery and biomolecular sensing

Abstract

DNA nanotechnology is an emerging field involving the fabrication of nanostructures with specified size and geometry using synthetic nucleic acid sequences. There are several advantages of DNA nanotechnology for biomedical applications. Firstly, DNA is a biocompatible material with low immunogenicity. Secondly, DNA strands can be assembled into structures with a pre-designed size and shape for optimal cell internalisation and prevention of renal clearance. Thirdly, DNA nanostructures can be functionalised with aptamers for enhanced and targeted cell delivery.

My PhD study bridges structural DNA nanotechnology and biomedical research, including in vitro diagnostic and targeted cell delivery in vitro and in vivo. I investigated three different tile-based nanostructures: two-dimensional DNA tiles, the DNA mazzocchio, and a DNA prism nanocalliper in this study. The DNA nanostructures were functionalised with protein-binding aptamers and with a split G-quadruplex DNAzyme for different applications.

My first aim was to develop a two-dimensional DNA tile integrated with a split DNAzyme as a sensing platform for the detection of short single-strand DNA and RNA molecules. The DNA tile forms 2D nanostructures with periodic gridding in the presence of target strands. Compared with a simple DNA nanotweezer, DNA tiles were shown to be able to amplify the detection signal and showed significant stability to nucleases.

My second aim was to functionalise the “doughnut”-like DNA mazzocchio with a nasopharyngeal carcinoma cell-specific aptamer for targeted cell delivery. DNA mazzocchio was assembled by self-replication of a subunit motif, rendering a wireframe DNA tile in a “doughnut”-like shape. Aptamer-functionalised DNA mazzocchio demonstrated superior cell-specific delivery efficacy when compared to a simple DNA tetrahedron. Also, the endocytosis route and intracellular fate of aptamer-functionalised DNA mazzocchio and tetrahedron in NPC cells were investigated. Surprising, the NPC aptamer changes the route for endocytosis of DNA nanostructures. Aptamer-functionalised DNA mazzocchio and tetrahedron were tested for in vivo tumour-targeted delivery, where mazzocchio demonstrated superior delivery efficacy by minimising renal filtration. A better understanding of the delivery process of DNA nanostructures inside cells would benefit drug delivery in the future.

My third aim was to design and construct a DNA prism nanocalliper through the single-stranded DNA tile principle. The DNA nanocalliper was used for the display of gold nanoparticles at a precise distance. The DNA nanocalliper was modified by bivalent aptamers for the diagnosis of SARS-Cov-2 viral nucleocapsid proteins at nanomolar sensitivity. In summary, different tile-based DNA nanostructures were functionalised with DNAzymes and protein-binding aptamers for biomedical applications. These static nanostructures were successfully converted into biosensors of nucleic acids, or into drug carriers with enhanced tumour cell targeting ability in vitro and in vivo. This work expands the application of DNA tile-based nanostructures. Aptamers are demonstrated as suitable “wingmen” for DNA nanostructures, and may facilitate future translation of DNA nanotechnology from bench to bedside.