title: Properties of short double-stranded RNAs carrying randomized base pairs: toward better controls for RNAi experiments.
abstract: Short interfering RNAs (siRNAs) are mediators of RNA interference (RNAi), a commonly used technique for selective down-regulation of target gene expression. Using an equimolar mixture of A, G, C, and U phosphoramidites during solid-phase synthesis, we introduced degenerate positions into RNA guide and passenger strands so that, when annealed, a large pool of distinct siRNA duplexes with randomized base pairs at defined sites was created. We assessed the randomization efficiency by deep sequencing one of the RNAs. All possible individual sequences were present in the pool with generally an excellent distribution of bases. Melting temperature analyses suggested that pools of randomized guide and passenger strands RNAs with up to eight degenerate positions annealed so that mismatched base-pairing was minimized. Transfections of randomized siRNAs (rnd-siRNAs) into cells led to inhibition of luciferase reporters by a miRNA-like mechanism when the seed regions of rnd-siRNA guide strands were devoid of degenerate positions. Furthermore, the mRNA levels of a select set of genes associated with siRNA off-target effects were measured and indicated that rnd-siRNAs with degenerate positions in the seed likely show typical non-sequence-specific effects, but not miRNA-like off-target effects. In the wake of recent reports showing the preponderance of miRNA-like off-target effects of siRNAs, our findings are of value for the design of a novel class of easily prepared and universally applicable negative siRNA controls.
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Julian A
authors.initials: JA
authors.lastname: Zagalak
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Mirjam
authors.initials: M
authors.lastname: Menzi
authors.affiliation: Computational Biology Group (CBG), Department of Biosystems Science and Engineering, ETH Zurich, CH-4058 Basel, Switzerland.
authors.forename: Fabian
authors.initials: F
authors.lastname: Schmich
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Hartmut
authors.initials: H
authors.lastname: Jahns
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Afzal M
authors.initials: AM
authors.lastname: Dogar
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Florian
authors.initials: F
authors.lastname: Wullschleger
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Harry
authors.initials: H
authors.lastname: Towbin
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Hall
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D015870:Gene Expression; D055785:Gene Knockdown Techniques / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection
chemical_list: D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering
keywords: RNAi; degenerate base; negative control; randomized base; siRNA