Fig. 6: Toehold switch regulator design (5-inputs AND gate) and characterization in cultured MDCK cells.
From: Circular single-stranded DNA as switchable vector for gene expression in mammalian cells
a The scheme illustration of the circular single-stranded gene expression regulator empowered by adding five single blocking strands and five toehold-switch trigger strands via TMSD reaction (5-input AND gate). b Assessment of seven toehold switches orthogonality consisting of block-input A, B, C, D, E, E1, and E2, in which block-input E1 and E2 only change toehold sequence on the basis of block-input E. Finput/Fblock represents EGFP fluorescence ratios of Css EGFP(+)-block with different input signal and Css EGFP(+)-block after 24 h of transfection in cultured MDCK cell. c The representative fluorescence images of cultured MDCK cells transfected with single Css EGFP(+), the blocked Css EGFP(+) with 5 blocking strands (5B) and the blocked Css EGFP( + )-5-input trigger strands (5B + 5-input), respectively. The images are representative of one of n = 3 biologically independent experiments; similar results were observed each time. Scale bar, 100 μm. d The representative flow cytometry GFP fluorescence analysis for five toehold switchable regulator compared to MDCK cell autofluorescence (blank) and a Css EGFP(+) positive control. Autofluorescence level was measured from MDCK cells not treated with Css EGFP(+). The flow analysis is representative of one of n = 3 biologically independent experiments; similar results were observed each time. e Fluorescence intensity of the circular single-stranded gene expression regulator (5-input AND gate). Fluorescence Norm. indicates that all fluorescence intensities were normalized to the fluorescence value of the corresponding mammalian cell transfected with the corresponding untreated Css EGFP(+). Lower fluorescence intensity was observed for 5B (p = 1.99 × 10−7) compared to untreated Css EGFP( + ). 5B + 5-input (p = 1.03 × 10−5) demonstrated higher fluorescence intensity than 5B. Data collected in b and e were quantified using flow cytometry and are presented as mean ± standard deviation (s.d.) for n = 3 biologically independent experiments, individual data points are overlaid in e, source data provided. Statistical analysis in e was performed using one-way ANOVA with Tukey’s multiple comparison (****p ≤ 0.0001, ns p > 0.05).
