Departments of Pharmacology and Biochemistry, UT Southwestern Medical Center at Dallas, Dallas, TX 75390, USA and Department of Medicinal Chemistry and Core Antisense Research, ISIS Pharmaceuticals, Carlsbad, CA 92010, USA.
Nucleic Acids Res. 2013 Nov;41(20):9570-83. doi: 10.1093/nar/gkt693. Epub 2013 Aug 9.
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.
单链沉默 RNA（ss-siRNA）为基因沉默提供了一种替代方法。ss-siRNA 结合了反义寡核苷酸的简单性和良好的生物分布性，以及通过 RNA 干扰（RNAi）实现的强大沉默效果。以前的研究报告称，针对突变等位基因中扩展的 CAG 重复序列的 ss-siRNA 能够有效且等位基因选择性地抑制人类亨廷顿蛋白的表达。Machado-Joseph 病的遗传原因是突变的ataxin-3，它也含有一个扩展的 CAG 重复序列。我们在这里证明，ss-siRNA 是ataxin-3 表达的等位基因选择性抑制剂，然后重新设计 ss-siRNA 以优化其选择性。我们发现，RNA 干扰相关和非 RNA 干扰相关机制都会通过阻断翻译或影响选择性剪接来影响基因表达。这些结果有四个广泛的意义：（i）ss-siRNA 并不总是与类似的 RNA 双链体表现出相同的行为；（ii）CAG 重复序列周围的序列会影响抗 CAG 寡核苷酸的等位基因选择性；（iii）ss-siRNA 可以通过多种机制发挥作用；以及（iv）可以使用化学修饰来优化 ss-siRNA 的性质，并提高其在药物发现中的潜力。
