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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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).
Structures of guide-RNA are correlated with RNAi. (PDF 33 kb)
RNase T1 probing of as-siRNA structures 4-7, 0-0, and 2-9. (PDF 45 kb)
Predicted mfe structures and accessibility profiles of local jagged-1 mRNA targets. (PDF 48 kb)
Classifying guide-RNA secondary structures. (PDF 55 kb)
Model describing the determination of RNA silencing by RNA secondary structures. (PDF 270 kb)
Target structure accessibility and RNAi. (PDF 67 kb)
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)
RNA secondary structure prediction: mfe vs. partition structures. (PDF 33 kb)
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