Design and self-assembly of DNA nano-architectures for bio-therapeutic and bio-technological applications

Abstract

DNA origami is a branch of structural DNA nanotechnology which traditionally involves self-assembly of a single stranded DNA plasmid (scaffold) into the target architectures, facilitated by shorter oligonucleotide sequences termed “helper” or “staple” strands. The objective of this study was to design two 3D DNA nano-architectures Nanoluc and NanomCherry with programmed instability. The architectures were to be assembled using both strands of covalently closed circular double stranded plasmids. The design of the two architectures was carried out in silico using the caDNAno square software. Parallel stranded DNA was rationally incorporated into the architectures’ designs to introduce weak points intended to confer instability. The effects of various annealing conditions on self-assembly of Nanoluc and NanomCherry were assessed by monitoring the emergence of fast migrating unique bands using agarose gel electrophoresis. Structures were subsequently eluted by electro-elution or homogenization then visualized by TEM. Gel analysis of Nanoluc and NanomCherry assembly mixes revealed staple directed structuring of the double stranded scaffold as evidenced by the emergence of fast migrating unique bands. Nanoluc’s most structured species were observed when assembly mixes containing 10:1 staple to scaffold in 60 % formamide were subjected to thermal treatment followed by immediate dialysis in formamide free folding buffer as confirmed by a TEM analysis. NanomCherry’s most structured species were observed after assembly mixes containing 10:1 staple to scaffold in 40 % or 60 % formamide were incubated at 90 ˚C for 15 minutes and rapidly cooled to 4 ˚C. Unfortunately we were unable to characterise the species in NanomCherry’s fast migrating unique bands due to time constraints. The study successfully demonstrated structuring of closed circular double stranded plasmid into 3D DNA nano-architectures. However further optimization of the design and self-assembly process is still required to achieve the target dimensions and enhanced yields.

Date of Award	May 2015
Original language	English
Supervisor	Colum Walsh (Supervisor)