Fig. 4. Enhancing gene expression through scaffold sequence design.
a 20HB design for the sc_EGFP5 structure included an external single-stranded loop to allow the ITR sequence to self-anneal and form the hairpin structure. 20HB design for the sc_EGFP6 scaffold included two external loops to expose the ITR binding domain, enabling the ITR hairpin staples to anneal. b, c Transfection efficiency and MFI in HEK293T cells seen for 20HB-exLP structures folded with sc_EGFP5 and sc_EGFP6. Higher transfection efficiencies and MFI were observed for 20HB-exLP objects folded with sc_EGFP5 (p = 0.0002, p = 7.99 × 10−5) and sc_EGFP6 (p = 0.0134, p = 4.6 × 10−8) compared to sc_EGFP1 20HB. In addition, 20HB-exLP folded with sc_EGFP6 demonstrated higher MFI than 20HB-exLP folded with sc_EGFP5 (p = 0.0169). Data collected in b and c were quantified using flow cytometry and are presented as mean ± s.d. for n = 3 biologically independent experiments, individual data points are overlaid, controls are unfolded scaffold and staple mixture ‘sc + st’ and scaffold only ‘sc’, 0.5 µg total DNA per condition, source data provided. Statistical analysis in b and c was performed using one-way ANOVA with Tukey’s multiple comparison, (*p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001, ns p > 0.05). d Representative epifluorescence microscopy images showing EGFP expression from cells transfected with DNA origami objects folded with sc_EGFP5 and sc_EGFP6 relative to sc_EGFP1. Images in the bottom row have been purposely contrast enhanced to reveal EGFP positive cells that have poor EGFP intensity in the sc_EGFP1 sample. The images are representative of one of n = 3 biologically independent experiments; similar results were observed each time. Scale bar 100 µm.