Rational optimization of siRNA to ensure strand bias in the interaction with the RNA-induced silencing complex

• Published in: Chemical communications (Cambridge, England) England), 2023-05, Vol.59 (42), p.6347-635
• Main Authors: Datta, Dhrubajyoti, Theile, Christopher S, Wassarman, Kelly, Qin, June, Racie, Tim, Schmidt, Karyn, Jiang, Yongfeng, Sigel, Rachel, Janas, Maja M, Egli, Martin, Manoharan, Muthiah
• Format: Article
• Language: English
• Subjects: Animals; Mice; Morpholinos - chemistry; Nucleotides; Optimization; Piperidine; RNA, Small Interfering - chemistry; RNA-Induced Silencing Complex - genetics; RNA-Induced Silencing Complex - metabolism
• ISSN: 1359-7345; 1364-548X
• DOI: 10.1039/d3cc01143g

To ensure specificity of small interfering RNAs (siRNAs), the antisense strand must be selected by the RNA-induced silencing complex (RISC). We have previously demonstrated that a 5′-morpholino-modified nucleotide at the 5′-end of the sense strand inhibits its interaction with RISC ensuring selection of the desired antisense strand. To improve this antagonizing binding property even further, a new set of morpholino-based analogues, Mo2 and Mo3 , and a piperidine analogue, Pip, were designed based on the known structure of Argonaute2, the slicer enzyme component of RISC. Sense strands of siRNAs were modified with these new analogues, and the siRNAs were evaluated in vitro and in mice for RNAi activity. Our data demonstrated that Mo2 is the best RISC inhibitor among the modifications tested and that it effectively mitigates sense strand-based off-target activity of siRNA. A new class of 5′-morpholino modified nucleoside building blocks was synthesized. Among them, Mo2 exhibited better off-target mitigation for RNAi compared to other analogues Pip and Mo3 and the previously reported morpholino derivative Mo1 .