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

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 selectio...

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Bibliographic Details
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
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Summary: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 .
ISSN:1359-7345
1364-548X
DOI:10.1039/d3cc01143g