ss-siRNAs allele selectively inhibit ataxin-3 expression: multiple mechanisms for an alternative gene silencing strategy

• Published in: Nucleic acids research Vol. 41; no. 20; pp. 9570 - 9583
• Main Authors: Liu, Jing, Yu, Dongbo, Aiba, Yuichiro, Pendergraff, Hannah, Swayze, Eric E, Lima, Walt F, Hu, Jiaxin, Prakash, Thazha P, Corey, David R
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.11.2013
• Subjects: Alleles; Alternative Splicing; Amino Acid Sequence; Ataxin-3; Cell Line; Humans; Molecular Sequence Data; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - genetics; Nuclear Proteins - chemistry; Nuclear Proteins - genetics; Repressor Proteins - chemistry; Repressor Proteins - genetics; RNA Interference; RNA, Small Interfering - chemistry; Synthetic Biology and Chemistry; Trinucleotide Repeats
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkt693

Single-stranded silencing RNAs (ss-siRNAs) provide an alternative approach to gene silencing. ss-siRNAs combine the simplicity and favorable biodistribution of antisense oligonucleotides with robust silencing through RNA interference (RNAi). Previous studies reported potent and allele-selective inhibition of human huntingtin expression by ss-siRNAs that target the expanded CAG repeats within the mutant allele. Mutant ataxin-3, the genetic cause of Machado-Joseph Disease, also contains an expanded CAG repeat. We demonstrate here that ss-siRNAs are allele-selective inhibitors of ataxin-3 expression and then redesign ss-siRNAs to optimize their selectivity. We find that both RNAi-related and non-RNAi-related mechanisms affect gene expression by either blocking translation or affecting alternative splicing. These results have four broad implications: (i) ss-siRNAs will not always behave similarly to analogous RNA duplexes; (ii) the sequences surrounding CAG repeats affect allele-selectivity of anti-CAG oligonucleotides; (iii) ss-siRNAs can function through multiple mechanisms and; and (iv) it is possible to use chemical modification to optimize ss-siRNA properties and improve their potential for drug discovery.