Whitehouse, William L.;
(2021)
Interfacing DNA Nanostructures with Biology.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
DNA nanostructures are powerful tools for synthetic biology with great promise as drug delivery devices. Their strength lies in the unique programmability offered by Watson-Crick base pairing and the ability to site-specifically incorporate bioactive components with nanoscale resolution. For example, lipid anchor cholesterols have been used to control binding of DNA nanostructures to synthetic and biological bilayer membranes. Additionally, various bioactive components have been attached to guide nanostructure interaction with eukaryotic cells. Yet, there is a lack of fundamental understanding of how cholesterol modification affects DNA nanostructure binding by cells and their uptake. Similarly, the endosome escape of DNA nanostructures remains a challenge and has not been achieved using programmed incorporation of escape-mediating components. This thesis explores both topics using two archetypal DNA nanostructures, a 6-duplex bundle and a rectangular DNA origami. The first is used to interrogate how individual cholesterol anchors affect membrane binding and uptake by cancer cells, using fluorescence microscopy as read-out and a nuclease digestion strategy. The second is used as a basis to explore programmed presentation of the cell-penetrating GALA peptide with known membrane destabilising properties to probe endosomal release. The findings reveal a clear correlation between cholesterol anchor number on the 6-duplex nanobundle, membrane binding and cell uptake kinetics. Fetal bovine serum is found to inhibit uptake but not membrane binding, suggesting an interplay between serum components, DNA nanostructures and the internalisation process in HeLa cells1. By comparison, presenting GALA peptides in defined number and position on a 50 x 60 nm DNA origami rectangle enhances binding to small unilamellar vesicles with the pH selectivity expected for GALA. The peptide also enhances origami-cell uptake as indicated by live-cell microscopy, with less certain results regarding endosome destabilisation and release. The insight on cholesterol-mediated cell uptake of DNA nanostructures as a function of protein environment will guide the design cell-specific nanostructures. Furthermore, programing endosomal release with cell-penetrating peptides on DNA nanostructures has potential implications for the development of nano therapeutic strategies.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Interfacing DNA Nanostructures with Biology |
| Event: | UCL |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2021. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Chemistry |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/10136201 |
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