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Design of siRNAs producing unstructured guide-RNAs results in improved RNA interference efficiency

Abstract

In RNA interference (RNAi), guide RNAs direct RNA-induced silencing complexes (RISC) to their mRNA targets, thus enabling the cleavage that leads to gene silencing. We describe a strong inverse correlation between the degree of guide-RNA secondary structure formation and gene silencing by small interfering (si)RNA. Unstructured guide strands mediate the strongest silencing whereas structures with base-paired ends are inactive. Thus, the availability of terminal nucleotides within guide structures determines the strength of silencing. A to G and C to U base exchanges, which involve wobble base-pairing with the target but preserve complementarity, turned inactive into active guide structures, thereby expanding the space of functional siRNAs. Previously observed base degenerations among mature micro (mi)RNAs together with the data presented here suggest a crucial role of the guide-RNA structures in miRNA action. The analysis of the effect of the secondary structures of guide-RNA sequences on RNAi efficiency provides a basis for better understanding RNA silencing pathways and improving the design of siRNAs.

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Acknowledgements

This work was supported by grants from the German Federal Ministry of Education and Research (BMBF) and the Senate of the city of Berlin 0313068C, 0313066F, 0313066C-11, SenBB3066C-11 (RNA Network) cosponsored by Chiron Corp. and amaxa biosystems and 01 GS 0413 (NGFN). S.R. was supported by a grant from the Boehringer Ingelheim Fonds. We thank T. Ritter for providing plasmid pBabe-Jagged-1. C.K. was partly affiliated with the STZ Nucleic Acids Design (http://www.stz-nad.com).

Author information

Competing interests

The authors C.K. and V.P. are avocationally affiliated to the Steinbeis Transfer Center for Nucleic Acids Design (STZ-NAD; http://www.stz-nad.com/). The STZ-NAD is an institution for technology transfer, which offers services around nucleic acids design. As full-time employess of the Max-Planck-Society, which is an institution of academic research, the authors ensure that declared interests did not influence the objectivity, integrity or perceived value of this article.

Correspondence to Volker Patzel.

Supplementary information

Supplementary Fig. 1

Structures of guide-RNA are correlated with RNAi. (PDF 33 kb)

Supplementary Fig. 2

RNase T1 probing of as-siRNA structures 4-7, 0-0, and 2-9. (PDF 45 kb)

Supplementary Fig. 3

Predicted mfe structures and accessibility profiles of local jagged-1 mRNA targets. (PDF 48 kb)

Supplementary Fig. 4

Classifying guide-RNA secondary structures. (PDF 55 kb)

Supplementary Fig. 5

Model describing the determination of RNA silencing by RNA secondary structures. (PDF 270 kb)

Supplementary Data

Target structure accessibility and RNAi. (PDF 67 kb)

Supplementary Discussion

A, GU-rich guide-RNA generated by base exchanges. B, Guide-RNA structures and siRNA duplex formation. C, Uracil versus Thymin in guide-siRNA 3’ overhangs. (PDF 60 kb)

Supplementary Note

RNA secondary structure prediction: mfe vs. partition structures. (PDF 33 kb)

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Further reading

Figure 1: In silico-selected jagged-1-directed guide-siRNA.
Figure 2: Activities of in silico-selected siRNA.
Figure 3: Programming active as-siRNA/guide-RNA structures by base exchanges.
Figure 4: SiRNA duplex structures determine Argonaute dependence of RISC.