Student Author(s)

Dianne S. Schwarz

UMMS Affiliation

Department of Biochemistry and Molecular Pharmacology; Department of Medicine, Division of Endocrinology and Metabolism

Publication Date

2006-09-13

Document Type

Article

Disciplines

Biochemistry, Biophysics, and Structural Biology | Genetics and Genomics | Life Sciences | Medicine and Health Sciences

Abstract

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. We systematically identified siRNAs that discriminate between the wild-type and mutant alleles of two disease genes: the human Cu, Zn superoxide dismutase (SOD1) gene, which contributes to the progression of hereditary amyotrophic lateral sclerosis through the gain of a toxic property, and the huntingtin (HTT) gene, which causes Huntington disease when its CAG-repeat region expands beyond approximately 35 repeats. Using cell-free RNAi reactions in Drosophila embryo lysate and reporter assays and microarray analysis of off-target effects in cultured human cells, we identified positions within an siRNA that are most sensitive to mismatches. We also show that purine:purine mismatches imbue an siRNA with greater discriminatory power than other types of base mismatches. siRNAs in which either a G:U wobble or a mismatch is located in the "seed" sequence, the specialized siRNA guide region responsible for target binding, displayed lower levels of selectivity than those in which the mismatch was located 3' to the seed; this region of an siRNA is critical for target cleavage but not siRNA binding. Our data suggest that siRNAs can be designed to discriminate between the wild-type and mutant alleles of many genes that differ by just a single nucleotide.

DOI of Published Version

10.1371/journal.pgen.0020140

Source

PLoS Genet. 2006 Sep 8;2(9):e140. Epub 2006 Jul 24. Link to article on publisher's site

Journal/Book/Conference Title

PLoS genetics

Related Resources

Link to Article in PubMed

PubMed ID

16965178

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