Multi-gene regulation using DNA-origami-AuNPs nanostructures in breast cancer cells

Gene regulation is a novel approach to cure a gene related disease. Thus, there is an ongoing effort to increase the efficiency of gene delivery. In this thesis, we developed a DNA based nanostructure to deliver gene regulation elements for the up-regulated genes in breast cancer as alternative to the currently used non-viral delivery systems. A tile shaped DNA origami nanostructure is constructed by including morpholino antisense oligonucleotides targeting the HER2, ERα, EGFR, and Ki-67 genes. Then, the sticky ends of the DNA origami nanostructures were hybridized to the complementary oligonucleotide sequence attached to gold nanoparticles (AuNPs) to enhance their cellular uptake. The constructed nanostructure was characterized using agarose gel electrophoresis, AFM, DLS and UV/Vis spectroscopy. Following the stability and toxicity analysis of the structure, the nanocarrier system was used for silencing of the mentioned genes in their overexpressing breast cancer cell lines. It was found that the antisense oligonucleotide embedded DNA origami-AuNPs structure was highly effective for inducing single and multiple-gene silencing in breast cancer cells. The prepared nanocarrier inhibited the expression of target genes approximately 50 per cent at 30 nM morpholino concentration. For multi-gene silencing, more than one morpholino oligos targeting different genes were embedded into DNA nanocarrier and the silencing efficiency increased to about 80 per cent by synergistic effect. After silencing of the target genes alone and in combination, the effects of the gene regulation on breast cancer cell proliferation and cell cycle phase distributions were investigated. It was showed that gene silencing using DNA origami-AuNPs nanostructures inhibited proliferation of the breast cancer cells and altered their cell cycle phase distributions. Silencing efficiency of DNA origami-AuNPs nanostructures was compared with commercial transfection agents and found to be more effective. Furthermore, DNA origami-AuNPs nanostructures did not affect viability of cancer and healthy cells, while the tested commercial systems were highly toxic. The proposed novel nanostructure provides an effective and biocompatible carrier for gene silencing studies.