Designing Ago2-specific siRNA/shRNA to Avoid Competition with Endogenous miRNAs.

Hongming Ma,Haoquan Wu,Junli Zhang

doi:10.1038/mtna.2014.27

Hongming Ma, Haoquan Wu + Show 1 more

Open Access

https://doi.org/10.1038/mtna.2014.27

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Abstract

Relatively large amounts of transfected siRNA can compete for Ago proteins and thus compromise endogenous miRNA function, potentially leading to toxicities. Here, we show that shRNA can also perturb endogenous miRNA function similarly. More importantly, we also show that the problem can be solved by designing shRNAs in the context of pre-miR-451 structure with completely complementary stem, which significantly improves the Ago2 specificity. This shRNA was shown to be Ago2-specific, and maintain target-silencing ability while avoiding competition with endogenous miRNAs by not competing for Agos 1, 3, and 4. We conclude that modified pre-miR-451 structure provides a general platform to design shRNAs that significantly reduce perturbation of miRNA function.

Highlights

In mammals, the canonical miRNA biogenesis pathway is initiated by the Drosha–DGCR8 complex, which processes long-primary miRNAs into short pre-miRNAs for further processing by Dicer into mature miRNA duplexes
In addition to different repression mechanisms, the loading of siRNA or miRNA can differ between Ago[2] and the other Ago proteins because Ago[2] can cleave the passenger strand, which is removed by C3PO;[7,8] while Ago[1, 3], and 4 cannot cleave the passenger strand, and must load siRNA or miRNA using the “bypass mechanism”
We tested the loading of siRNA or miRNA-like duplexes into different Ago proteins in these cells and showed that all four Ago proteins can load both siRNA and miRNA structured oligos to generate active RNAinduced silencing complex (RISC)

Summary

Introduction

The canonical miRNA biogenesis pathway is initiated by the Drosha–DGCR8 complex, which processes long-primary miRNAs (pri-miRNAs) into short pre-miRNAs for further processing by Dicer into mature miRNA duplexes One or both strands of the duplex are loaded into the RNAinduced silencing complex (RISC) to suppress target genes.[1,2] This endogenous miRNA-processing machinery is the basis for why exogenously introduced siRNA/shRNA can function in cells. SiRNAs and shRNAs mimic miRNA biogenesis intermediates at different stages—siRNAs are mainly mimics of miRNA duplexes while conventional shRNAs are mimics of pre-miRNAs. One or both strands of the duplex are loaded into the RNAinduced silencing complex (RISC) to suppress target genes.[1,2] This endogenous miRNA-processing machinery is the basis for why exogenously introduced siRNA/shRNA can function in cells. In addition to different repression mechanisms, the loading of siRNA or miRNA can differ between Ago[2] and the other Ago proteins because Ago[2] can cleave the passenger strand, which is removed by C3PO (component 3 promoter of RISC, a complex of Translin and Trax);[7,8] while Ago[1, 3], and 4 cannot cleave the passenger strand, and must load siRNA or miRNA using the “bypass mechanism”.9 Ago[2] is distinct from the other three Ago proteins in its slicer function

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