Assembly and intracellular delivery methods for nucleic acid nanotechnology

Thesis

The field of nucleic acid nanotechnology has mainly taken DNA and RNA out of their biologicalvcontext and uses their properties to revolutionise the control of molecular self-assembly; but can we bring this technology back into the cell? Since their inception, nucleic acid nanostructures have developed into almost any shape imaginable, but one application area for these constructs that has not been explored as much — despite the advantages in size, biocompatibility and the programmability of these constructs— is at the interface with biology. Challenges preventing the breakthrough of this field into more intracellular applications involve improving the cellular uptake of the assembled constructs and, once inside the cell, protecting these nanostructures from nuclease degradation or unfolding. Overcoming these issues with new intracellular delivery methods or self-assembly designs that allow for de novo construction within the cell would open up a plethora of applications in diagnostics, therapeutics, drug delivery, as well as providing programmable tools for studying molecular biology within the cell itself.

This thesis investigates three approaches for designing intracellular nucleic acid nanostructures. The first strategy presented is a new isothermal single-stranded assembly method, inspired by knitting, for designing nucleic acid nanostructures inside the cell. I show the characterisation of both a single-knit and multi-knit structure, through both computational modelling as well as experimental analysis. The resulting knitted constructs were primarily focused on in vitro replicable DNA systems to allow for ease in characterisation, although I also delve into the concept as a co-transcriptionally folded RNA method. Using atomic force microscopy, I was able to visualise the rolling circle amplified multi-knitted constructs. Taking inspiration from knitting, this assembly technique has the potential to be used for further complex designs and patterns. I then explore alternative approaches towards bringing nucleic acid nanostructures into the cell. This includes the use of confinement as a new method for delivering asymmetrical DNA origami cryoelectron tomography tags into mammalian cells through the creation of transient pores in the membrane to allow for the cellular uptake and targeting of the DNA nanostructures. Finally, I demonstrate three variations of bistable DNA hairpins to show reconfiguration kinetics that could be used in the design of future intracellular nucleic acid nanostructures. With these three projects, I was able to demonstrate a variety of in vivo implementation of DNA nanostructures, which can inspire future exploration of this promising field of intracellular nucleic acid nanostructures.

Authors: Fan, CH

• DOI: 10.5287/ora-2rd5d4rgx
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: cell confinement; isothermal single-stranded assembly; DNA knitting; DNA nanotechnology
• Subjects: Physics; Synthetic biology; Biotechnology; Biophysics