Valenzuela Rachel A P, Onizuka Kazumitsu, Ball-Jones Alexi A, Hu Tiannan, Suter Scott R, Beal Peter A
Department of Chemistry, University of California, One Shields Avenue, Davis, CA, 95616, USA.
Chembiochem. 2016 Dec 14;17(24):2340-2345. doi: 10.1002/cbic.201600453. Epub 2016 Nov 7.
Short interfering RNA (siRNA)-triggered gene knockdown through the RNA interference (RNAi) pathway is widely used to study gene function, and siRNA-based therapeutics are in development. However, as the guide strand of an siRNA can function like a natural microRNA (miRNA), siRNAs often repress hundreds of off-target transcripts with complementarity only to the seed region (nucleotides 2-8) of the guide strand. Here, we describe novel guide strand 3'-end modifications derived from 1-ethynylribose (1-ER) and copper-catalyzed azide-alkyne cycloaddition reactions and evaluate their impact on target versus miRNA-like off-target knockdown. Surprisingly, when positioned at the guide strand 3'-end, the parent 1-ER modification substantially reduced off-target knockdown while having no measurable effect on on-target knockdown potency. In addition, these modifications were shown to modulate siRNA affinity for the hAgo2 PAZ domain. However, the change in PAZ domain binding affinity was not sufficient to predict the modification's effect on miRNA-like off targeting.
通过RNA干扰（RNAi）途径由短干扰RNA（siRNA）引发的基因敲低被广泛用于研究基因功能，基于siRNA的疗法也正在开发中。然而，由于siRNA的引导链可以像天然微小RNA（miRNA）一样发挥作用，siRNAs通常会抑制数百种仅与引导链的种子区域（核苷酸2-8）互补的脱靶转录本。在此，我们描述了源自1-乙炔核糖（1-ER）和铜催化的叠氮化物-炔烃环加成反应的新型引导链3'端修饰，并评估了它们对靶标与miRNA样脱靶敲低的影响。令人惊讶的是，当位于引导链3'端时，母体1-ER修饰显著降低了脱靶敲低，而对靶标敲低效力没有可测量的影响。此外，这些修饰被证明可调节siRNA对hAgo2 PAZ结构域的亲和力。然而，PAZ结构域结合亲和力的变化不足以预测该修饰对miRNA样脱靶的影响。
