Synthetic DNA and RNA origami scaffolds, towards in vitro and in vivo applications

Abstract

Nucleic acid self-assembly is a branch of nanotechnology that consists in the use of DNA and RNA, not only as genetic material, but also as structural medium. In the last 30 years this technique developed from the simplest structures into more refined constructions that could require thousands of interacting sequences. DNA origami were presented by Rothemund as the evolution of DNA assemblies. Origami are nanostructures formed by a long single strand, the scaffold, held together by many shorter oligonucleotides, the staples. Later research showed how to functionalize the origami staples increasing the applicability. Bacteriophage genomes were used as convenient source of single stranded DNA scaffold. Today the potential biotechnological applications require to be bio-orthogonal and easily transferable from academic research to industrial ones. To solve these problems we can rely on the powerful tools of synthetic biology. I focused on the synthesis, analysis and application of synthetic scaffolds based on the combinatorial De Bruijn (DB) sequence. DB scaffolds are designed to be bio-orthogonal, uniquely addressable and thermodynamically optimized. I demonstrated their folding efficiency in DNA origami and RNA/DNA hybrid origami. DB scaffolds were analyzed in vivo in E: coli to asses their bio-orthogonality. Finally I designed a research tool to improve our knowledge on origami folding dynamics. The system includes functionalized origami encapsulated in giant unilamellar vesicles. The system allows the analysis of the origami using high throughput technologies, and can simulate a simple cell-like environment facilitating the intermediate steps towards the in vivo origami folding. With my interdisciplinary approach I contributed to the advancement of nucleic acid origami technology, taking advantage of tools and techniques from synthetic biology, origin of life, bioinformatics along with more classical scientific disciplines.