Development of nucleic acid-based nanoplatforms for applications in disease diagnostics and the study of nanomaterials-protein interactions
Creators: name: Wang, Ye
Publisher: name: Imperial College London
Year: 2018

description: The impact of nucleic acids on scientific and medical progress has been enormous. DNA is the blueprint for structure and function from the level of individual cells up to whole organisms, and various forms of RNA are all involved in the regulation of genetic information. The identification of specific nucleic acid sequences and/or their level of expression provide key information for the molecular identity and organism functional state. This is very useful in areas like biomedicine. Nucleic acids are also versatile structural materials at the nanoscale. The precise recognition pattern of the Watson-Crick base pairs makes them not only successful as genetic materials, but also capable of directing the assembly of highly structured materials with unique nanoscale features. Nucleic acids, either alone or in combination with other materials, have been used to create a number of nanoscale structures and devices that perform actively in an engineered environment. In this thesis, research towards nucleic acids as a fundamental tool for both diagnostic purpose and structural applications is described. The thesis mainly consists of two studies. The first study is focused on nucleic acid-based biosensing using fluorescent quantum dots (QDs) as donors in a fluorescence resonance energy transfer (FRET) assay for addressing the analytical needs for DNA or RNA detection. The optimization of protocols for synthesizing QD-DNA constructs and their applications in biosensing assays are discussed. A highly sensitive and specific microRNA (miRNA) assay was then developed by the integration of the QD-DNA constructs and an isothermal enzyme-mediated target recycling step, with a detection limit of 42 fM and excellent selectivity for miR-148 versus base-mismatched sequences and other miRNAs. This proposed method was successfully employed for detection of miR-21 using an alternative FRET pair, which was compared to qRT-PCR for the quantitative analysis of miR-21 in biological samples. The second study is focused on construction and characterization of nucleic acid-based hierarchical porous nanostructures and presents a concept for exploiting such constructs as scaffolds for enzyme immobilization and activity studies. Based on rolling circle replication (RCR), DNA or RNA structures with flower-shaped morphologies were synthesized by interactions between inorganic magnesium pyrophosphate (Mg2PPi) crystals and DNA or RNA strands in a time-dependent manner. Focusing on RNA-based structures, various characterization techniques were applied to understand the composition and structure of the RNA particles, and different methods were taken for immobilizing protein or enzymes onto RNA particle. As a proof of principle study, β-galactosidase (β-gal) and horseradish peroxidase (HRP) enzymes were coupled to the RNA particles, and both exhibited enhanced enzymatic activity and improved stability in comparison to free enzymes. This RNA-based biomaterial provides a model to develop a wide range of biocatalysts and offers the promise of potent protein loading and delivery system for biomedical applications.