Fig. 1: The primary screen for optimal siRNA sequence yields potent compounds with moderate discriminating power. | Nature Communications

Fig. 1: The primary screen for optimal siRNA sequence yields potent compounds with moderate discriminating power.

From: Chemical engineering of therapeutic siRNAs for allele-specific gene silencing in Huntington’s disease models

Fig. 1

a siRNA structure and chemical modification pattern used in screening. b The primary screen identifies the most favorable positions of the SNP enabling single nucleotide discrimination for targeting of SNP site rs362273 (highlighted in red). Compounds were tested using a dual-luciferase reporter assay system in HeLa cells. The (psiCheck) reporter plasmids contain a 40 mer region of huntingtin, including the target (A) SNP (black), and non-target (G) isoform (red). Cells were treated for 72 h at 1.5 µM of siRNA. A panel of siRNA sequences were synthesized in a cholesterol-conjugated scaffold with phosphorothioate and alternating 2′-F and 2′-OMe backbone modifications. By walking the siRNA sequence around SNP site rs362273, we find multiple compounds with varying degrees of efficacy and discrimination; n = 3 wells/treatment. c 7-point dose-response shows that siRNAs with the SNP site in positions 4 (SNP4) and 6 (SNP6) generate 20-fold and 7-fold allelic discrimination, respectively, with a high degree of efficacy; n = 2 wells/treatment. All data are presented as mean ± standard deviation. Source data are provided as a source data file.

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