Quantitative evaluation of first, second, and third generation hairpin systems reveals the limit of mammalian vector-based RNAi

RNA Biology
Colin WatanabeBenjamin Haley

Abstract

Incorporating miRNA-like features into vector-based hairpin scaffolds has been shown to augment small RNA processing and RNAi efficiency. Therefore, defining an optimal, native hairpin context may obviate a need for hairpin-specific targeting design schemes, which confound the movement of functional siRNAs into shRNA/artificial miRNA backbones, or large-scale screens to identify efficacious sequences. Thus, we used quantitative cell-based assays to compare separate third generation artificial miRNA systems, miR-E (based on miR-30a) and miR-3G (based on miR-16-2 and first described in this study) to widely-adopted, first and second generation formats in both Pol-II and Pol-III expression vector contexts. Despite their unique structures and strandedness, and in contrast to first and second-generation RNAi triggers, the third generation formats operated with remarkable similarity to one another, and strong silencing was observed with a significant fraction of the evaluated target sequences within either promoter context. By pairing an established siRNA design algorithm with the third generation vectors we could readily identify targeting sequences that matched or exceeded the potency of those discovered through large-scale sensor-...Continue Reading

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Jan 13, 2018·Human Gene Therapy Methods·Felix F AdamsAdrian Schwarzer
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May 23, 2020·Cancer Cell·Benjamin Haley, Filip Roudnicky
Sep 8, 2021·Cancer Gene Therapy·Mahsa SalianiMohammad Reza Ahmadian

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