Fig. 2: Optimization of gene expression through staple design and scaffold orientation.
From: Gene-encoding DNA origami for mammalian cell expression
Scaffold routing and schematics of unfolded scaffold for 20HB-LPv2 and 20HB-Circ designs, a and b respectively. 20HB-LPv2 incorporates two continuous 154-mer staples (pink), and 20HB-Circ design has been routed so that the 200-mer staple (pink), which acts as a splint, brings together the 5’ start of the CMV, and the 3’ of the polyA. c TEM micrographs of 20HB-LPv2 and 20HB-Circ, scale bar 100 nm. d Transfection efficiency into HEK293T cells via electroporation revealed higher transfection efficiency for samples 20HB-LP (p = 0.00004), 20HB-LPv2 (p = 1.87 × 10−7) and 20HB-Circ (p = 1.82 × 10−7), when compared to the standard 20HB. e Scaffold used encoded for the ‘coding strand’, where the expression cassette is present in the 5’ to 3’ direction (sc_EGFP1, upper panel). Scaffold encoding for the reverse complementary sequence of the expression cassette, thus the ‘template strand’ (sc_EGFP2, lower panel) was designed and produced. f Transfection efficiency into HEK293T cells by electroporation with either sc_EGFP2 scaffold + staple mixture, scaffold only, or folded 20HB objects. Electroporation with the template strand, sc_EGFP2, demonstrated higher transfection efficiencies for sc + st (p = 0.0051) and sc (p = 0.0018) conditions, compared to sc_EGFP1. g EGFP mean fluorescence intensity (MFI) for 20HB, 20HB-LPv2 and 20HB-Circ structures folded with either the coding or the template strand as the scaffold, transfected into HEK293T cells. Structure 20HB-LPv2 demonstrated higher MFI when folded with the template strand (p = 0.0388). Data collected in d, f and g 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 d was performed using one-way ANOVA with Tukey’s multiple comparison, while statistical analysis for f and g was performed using two-tailed Student’s t-test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns p > 0.05).
