Designing siRNA that distinguish between genes that differ by a single nucleotide

Small interfering RNAs (siRNAs), the guides that direct RNA interference (RNAi), provide a powerful tool to reduce the expression of a single gene in human cells. Ideally, dominant, gain-of-function human diseases could be treated using siRNAs that specifically silence the mutant disease allele, while leaving expression of the wild-type allele unperturbed. Previous reports suggest that siRNAs can be designed with single-nucleotide specificity, but no rational basis for the design of siRNAs with single nucleotide discrimination has been proposed. Keywords: siRNA, single-nucleotide discrimination, SOD1, allele-specific We transfected the siRNAs into cultured human HeLa cells at 100 nM final concentration. Amplified cRNA from siRNA-transfected cells was hybridized against cRNA from mock-transfected cells (treated with transfection reagent in the absence of RNA duplex). Ratio hybridizations were performed with fluorescent label reversal to eliminate dye bias.