Fig. 5: Toehold switch regulator design (2-input AND gate) and characterization in cultured MDCK cells.
From: Circular single-stranded DNA as switchable vector for gene expression in mammalian cells
The design (a) and fluorescence intensities (b) of Css EGFP(+) regulator (1-input YES) empowered by adding a single block strand and a toehold-switch trigger strand to CMV or EGFP region via TMSD reaction. Lower fluorescence intensities were observed for Toe-CB(M1) (p = 2.0 × 10−9) and Toe-EB(M4) (p = 1.2 × 10−9) compared to untreated Css EGFP(+). In addition, Toe-CB(M1)+trigger (p = 6.29 × 10−7) and Toe-EB(M1)+trigger (p = 2.57 × 10−7) demonstrated higher fluorescence intensities than Toe-CB(M1) and Toe-EB(M1), respectively. The design (c) and fluorescence intensities (d) of Css EGFP(+) regulator (2-input AND gate) empowered by adding two single blocking strands and two toehold-switch trigger strands to CMV and EGFP region via TMSD reaction. Lower fluorescence intensity was observed for doubly block (2B) (p = 1.11 × 10−5) compared to untreated Css EGFP(+). In addition, 2B + 2-input (p = 1.82 × 10−4) demonstrated higher fluorescence intensity than 2B. The design (e) and fluorescence intensities (f) of Css mCherry(+) regulator (2-input AND gate) empowered by adding two single blocking strands and two toehold-switch trigger strands to CMV and non-coding region via TMSD reaction. Lower fluorescence intensity was observed for doubly block (2B) (p = 8.45 × 10−8) compared to untreated Css mCherry(+). 2B + 1-input (p = 0.0155) demonstrated higher fluorescence intensity than 2B, while 2B + 2-input (p = 2.48 × 10−5, p = 0.0008) demonstrated higher fluorescence intensities compared to 2B and 2B + 1-input, respectively. Data collected in b, d and f 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, source data provided. In b, d and f, Fluorescence Norm. indicates that all fluorescence intensities were normalized to the fluorescence value of the corresponding mammalian cell transfected with the corresponding untreated Css DNA. Statistical analysis in b, d and f was performed using one-way ANOVA with Tukey’s multiple comparison (*p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001, ns p > 0.05).
