• Anne-Kathrine Kure Larsen
4. semester, Nanobioteknologi (cand.polyt.), Kandidat (Kandidatuddannelse)
The overall goal of this project was to investigate DNA origami as a novel method for efficient gene delivery and expression in mammalian cells. A plasmid encoding Cas9 and EGFP was used as a basis for DNA origami scaffold creation. PCR was utilized to amplify a target region in plasmid pCas9_GFP necessary for expression. PCRs revealed a template highly resistant to complete denaturation due to its GC-rich chicken β-actin promoter and chimeric intron region. A hairpin formation is suggested to prevent full-length amplification of the template as well. The double-stranded target region in plasmid lentiCas9-EGFP was successfully created
instead. DNA origami modelling was performed, and two prospective designs are suggested. Simulations revealed apparent stability of the two structures in physiological conditions. Annealed oligo constructs and a model origami were used for initial studies of FRET detection of folded/unfolded states of DNA origami. The results hereof revealed a FRET signal for the annealed oligos, while further experimentation is needed to make a clear conclusion for denatured oligos and DNA origami.
Udgivelsesdato3 jun. 2020
Antal sider107
ID: 333521788