5154–5164 Nucleic Acids Research, 2007, Vol. 35, No. 15 doi:10.1093/nar/gkm543 Published online 26 July 2007 Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC Daniela Castanotto1, Kumi Sakurai1,2, Robert Lingeman 3 , Haitang Li1, Louise Shively3, Lars Aagaard1, Harris Soifer1, Anne Gatignol4, Arthur Riggs2,3 and John J. Rossi1,2,* 1Division of Molecular Biology, Beckman Research Institute of the City of Hope, 2Graduate School of Biological Sciences, 3Division of Biology, Beckman Research Institute of the City of Hope and 4Virus-cell interactions laboratory, Lady Davis Institute for Medical Research, McGill University, Montre´ al, Canada Received March 22, 2007; Revised June 14, 2007; Accepted July 4, 2007 ABSTRACT Despite the great potential of RNAi, ectopic expres- sion of shRNA or siRNAs holds the inherent risk of competition for critical RNAi components, thus altering the regulatory functions of some cellular microRNAs. In addition, specific siRNA sequences can potentially hinder incorporation of other siRNAs when used in a combinatorial approach. We show that both synthetic siRNAs and expressed shRNAs compete against each other and with the endogen- ous microRNAs for transport and for incorporation into the RNA induced silencing complex (RISC). The same siRNA sequences do not display competition when expressed from a microRNA backbone. We also show that TAR RNA binding protein (TRBP) is one of the sensors for selection and incorporation of the guide sequence of interfering RNAs. These findings reveal that combinatorial siRNA approaches can be problematic and have important implications for the methodology of expression and use of therapeutic interfering RNAs. INTRODUCTION The phenomenon of RNA interference was ﬁrst observed almost a decade ago (1) and seems to have evolved as a defense mechanism against foreign double-stranded RNA. It is triggered by short RNA duplexes ( 21–23 nt in length), which are processed from longer double-stranded RNA transcripts (2,3). Two major classes of small double- stranded RNA molecules have thus far been identiﬁed: small interfering RNAs (siRNAs) and microRNAs (miRNAs). SiRNAs are fully complementary double- stranded molecules while miRNAs originate as duplexes that have bulges. Both classes of double-stranded RNAs assemble in the RNA induced silencing complex (RISC), which contains key proteins for the processing and functioning of double-stranded RNAs in RNAi. A member of the Argonaute family, Ago-2, has been identiﬁed as the catalytic core of this complex (4–8). Since their discovery, the use of siRNAs has quickly widened to diverse areas of research and as potential therapeutic agents (9). High-throughput analysis based on the use of siRNA libraries is also revolutionizing the ﬁeld of functional genomics (9–11). However, RNAi is an important component of endogenous cellular processes (12) involved in post-transcriptional regulation of endo- genous gene expression (13,14) as well as antiviral protection (15–18). Interfering with the endogenous functions of RNAi could lead to severe toxicity as recently demonstrated (19). The key component for the cellular export of shRNAs and microRNAs is the nuclear karyopherin Exportin-5 (20–23). In the presence of Ran-GTP, Exportin-5 binds to both shRNAs and microRNAs transporting them from the nucleus to the cytoplasm. Exportin-5 is a saturable transport pathway so the excessive production of small interfering RNAs could result in a decrease of cellular miRNA function (19,24). This export function is not required for the activity of synthetic siRNAs (20). In this work, we show that the competition between shRNAs with cellular miRNAs is a general phenomenon that also takes place with synthetic siRNA sequences. The ability to compete varies with the sequence and does not depend solely on the saturation of Exportin-5. We show that siRNAs, which do not depend on Exportin-5 for their transport to the cytoplasm, retain their ability to compete against shRNAs and microRNAs. Ectopic expression of Exportin-5 only partially relieves the competition between shRNAs and endogenous miRNAs or exogenous siRNAs. *To whom correspondence should be addressed. Tel: +626 301 8360; Fax: +626 301 8271; Email: jrossi@bricoh.edu 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.