Local RNA Target Structure Influences siRNA Efficacy: A Systematic Global Analysis

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The efficiency with which small interfering RNAs (siRNAs) down-regulate specific gene expression in living cells is variable and a number of sequence-governed, biochemical parameters of the siRNA duplex have been proposed for the design of an efficient siRNA. Some of these parameters have been clearly identified to influence the assembly of the RNA-induced silencing complex (RISC), or to favour the sequence preferences of the RISC endonuclease. For other parameters, it is difficult to ascertain whether the influence is a determinant of the siRNA per se, or a determinant of the target RNA, especially its local structural characteristics. In order to gain an insight into the effects of local target structure on the biological activity of siRNA, we have used large sets of siRNAs directed against local targets of the mRNAs of ICAM-1 and survivin. Target structures were classified as accessible or inaccessible using an original, iterative computational approach and by experimental RNase H mapping. The effectiveness of siRNA was characterized by measuring the IC50 values in cell culture and the maximal extent of target suppression. Mean IC50 values were tenfold lower for accessible local target sites, with respect to inaccessible ones. Mean maximal target suppression was improved. These data illustrate that local target structure does, indeed, influence the activity of siRNA. We suggest that local target screening can significantly improve the hit rate in the design of biologically active siRNAs.

Introduction

RNA interference is an evolutionarily conserved process in which short regions of double-stranded RNA are produced endogenously from larger precursors and induce the post-transcriptional inhibition of the expression of specific genes.1, 2 The experimental introduction of such small interfering RNA (siRNA) into eukaryotic cells is rapidly becoming one of the most important new tools in functional genomics and holds great potential as a therapeutic strategy.3

The efficiency with which siRNA down-regulates specific gene expression in living cells is highly variable. Indeed, large-scale screens of a number of mRNAs have highlighted extensive positional effects, and some siRNAs show limited efficiency.4, 5, 6 This suggests that the efficacy of a specific siRNA species is influenced by intrinsic properties of the siRNA itself, by intrinsic properties of the target system (such as target RNA structure) or by a combination of both. A number of thermodynamic and structural characteristics of the siRNA moiety have been suggested to contribute to the effectiveness of siRNA.7, 8 Recent studies have led to the establishment of a list of eight such parameters that, when incorporated into a rational design algorithm, permit an increase in the probability of selecting an effective siRNA (i.e. one that is capable of down-regulating gene expression >50%.6 This leads to the suggestion that siRNA could be effective independently of its target sequence. However, although it is becoming clear that certain of the biochemical characteristics of the siRNA duplex influence the probability of the antisense siRNA strand (asRNAi) being recruited by the RNA-induced silencing complex (RISC),7, 8 it is not yet possible to establish for others whether the critical parameter affected is siRNA-dependent (i.e. RISC formation), target-dependent (i.e. sequence- or structure-dependent), or asRNAi/target duplex-dependent (i.e. substrate for endonuclease activity).

A number of reports have suggested that the selection of local target sequences is critical,4, 5, 9 as is the local structural target accessibility.4, 5, 9, 10, 11 The complex secondary structure of mRNAs, however, is not amenable to accurate modelling,12, 13, 14, 15 making it difficult to correlate siRNA efficacy to target structure. By narrowing the definition of secondary structure to two broad categories, accessible or non-accessible (vis à vis to Watson–Crick binding) and using an established, iterative computational analysis,16, 17 relevant structural information can be gleaned from primary sequence. This kind of theoretical approach has been demonstrated to give results that are highly consistent with structural probing of RNA in the presence of cellular extracts,18 suggesting that it represents a valid basis to study the relationship between target accessibility and efficacy of siRNA.

In order to better define the role of local target structure in siRNA efficacy, we have estimated IC50 values for siRNAs targeted to regions defined, by this computational analysis, as structurally accessible, or inaccessible, for two biologically distant target systems ICAM-1 and survivin. In addition, for the survivin transcript, we have used combinatorial oligonucleotide–RNase H mapping to confirm computed accessibility. We demonstrate that siRNA targeting putative accessible sites have a higher probability of exhibiting low IC50 and show greater maximal inhibition than those targeting putative inaccessible sites. Our data are in line with the accompanying study by Schubert et al. (accompanying paper) using intentionally designed target structures to study the relationship between local accessibility and efficacy.31 This suggests that target structure does, indeed, influence siRNA activity.

Section snippets

The ICAM-1 and survivin targets: structural analysis and selection of local target sites of mRNA

The expression of ICAM-1 in ECV304 cells and the expression of survivin in K562 cells represent two independent biological systems for this study. For both systems, experimental protocols, including cell culture conditions and transfection protocols, have been optimized. Target sites along the ICAM-1 and survivin mRNAs were analysed by a systematic computational calculation of local RNA secondary structures and classified according to their structural accessibility as described.11, 16 Briefly,

Discussion

It is commonly agreed that not all siRNA species designed against a given target are equally effective, and a number of models have been postulated to explain this observation. It has often been assumed that the biological effectiveness of siRNA is independent of characteristics of the target mRNA and is influenced solely by characteristics intrinsic to individual siRNAs. Thus, the first guidelines established, in order to design efficient siRNAs, put greater emphasis on siRNA sequence per se

Oligonucleotides

All oligoribonucleotides used here were synthesized with two 3′-deoxythymidine nucleotides and quality assured by PAGE purification and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Eurogentec, Seraing, Belgium). Prior to use, oligonucleotides were quantified by UV absorption spectroscopy and their integrity was controlled on denaturing, 12% (w/v) polyacrylamide gels followed by staining with Stains-All (Sigma-Aldrich, Deisenhofen, Germany). The

Acknowledgements

We thank the Medical Faculty of the University of Lübeck for financial support within the program FSO to R.K.K.F. and to G.S., and the Association pour la Recherche sur le Cancer to I.R. and to B.L.

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