Fig. 4: Various blocking strands for suppressing gene expression of Css DNA.

a Schematic showing single fused strands (48 nt) (each 2.5 pmol) which contain two complementary sequences shown in Fig. 3c were separately added to Css DNA (0.5 pmol). b The corresponding products in a were transfected into cultured MDCK cells (24 h) to obtain corresponding fluorescence intensities. c Various blocking strands in CMV promoter (yellow) termed “CMV blocks (CB)” or non-coding region (black) termed “non-conding blocks (NB)” were separately added to Css EGFP(+) and Css mCherry(+), respectively. d The corresponding hybridization products in c were transfected into cultured MDCK cells for 24 h and to record corresponding mean fluorescence intensities. The dots in green and red are the expression efficiencies of Css EGFP(+) and Css mCherry(+), respectively, affected by the corresponding blocking strand. e The construction of different complicated structures on EGFP region of Css EGFP(+) by adding various number staple strands (1, 7, 28, and 40 staples, respectively). f The expression efficiency analysis of cultured MDCK cells transfected with the corresponding products in e. Lower fluorescence intensity was observed for 1 staple (p = 1.67 × 10⁻⁷) compared to untreated Css EGFP(+). In addition, 28 staples (p = 1.66 × 10⁻⁴) and 40 staples (p = 5.74 × 10⁻⁵) demonstrated lower fluorescence intensities than 1 staple. 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 for b and f, 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 untreated Css EGFP(+). Statistical analysis in f was performed using one-way ANOVA with Tukey’s multiple comparison (***p ≤ 0.001, ****p ≤ 0.0001, ns p > 0.05).