OptiRNAi, an RNAi design tool

Abstract

RNA interference (RNAi), a recently developed reverse genetics tool, has many advantages compared to traditional gene knockout methods. Appropriate selection of double stranded RNAs identical to a specific region(s) of the target gene is critical for the successful implementation of this technology. Recently, Elbashir et al. [Methods 26 (2002) 199] has established empirical criteria for siRNA sequence selection that significantly improved the success rate for RNAi attempts. We have developed OptiRNAi, a computational tool, which uses the Elbashir et al. criteria to predict appropriate target sequences for siRNA production. Specificity of these siRNAs for the target of interest can then be assessed by the investigator using the embedded Blast search engine optimized for RNAi design. Thus, OptiRNAi is an efficient and user friendly tool for RNAi design based on criteria that are more stringent than other available tools.

Introduction

In 1998, Fire et al. [10] discovered RNA interference (RNAi), a sequence specific gene silencing mechanism in the nematode, Caenorhabditis elegans. RNAi is a multistep process involving the generation of a large double stranded RNA (dsRNA) which is cleaved into small interfering RNAs (siRNAs) in vivo by the RNase III endonuclease, Dicer. These siRNAs downregulate target gene expression by forming an RNA induced silencing complex (RISC) with cellular proteins. RISC promotes the degradation of mRNAs containing sequences similar to the siRNA component and also may silence the target gene by recruitment of chromatin remodeling enzymes [5], [12], [20], [23].

Initially, RNAi was used to investigate gene function in plants, worms and Drosophila [4], [10], [19]. Its application to mammalian systems appeared limited at first since dsRNAs longer than 30 nucleotides are recognized as foreign, potentially virally derived nucleic acids by dsRNA activated protein kinase (PKR). Activated PKR significantly alters cell metabolism which often results in apoptosis independent of the RNA sequence [22]. Recently, gene specific RNAi effects have been reported in mammalian cells when dsRNA length is limited to 21–23 nucleotides [8]. The specificity of these siRNAs appeared to be high in a human kidney cell line (HEK) as assayed by microarray analysis [3], but similar analysis in HeLa cells suggested that siRNA mediated gene silencing may affect other genes sharing sequence similarity with the target [15].

RNAi has been successfully introduced into mammalian cells by both direct transfection of synthetic siRNAs [7] and by the transfection of siRNA expression vectors transcribed by RNA polymerase III [21]. It is clear that appropriate design of the siRNA molecule is essential for the success of RNAi mediated gene silencing. Recently, Elbashir et al. has established empirical criteria for siRNA design [7]. Since these criteria are cumbersome to assess manually, we have developed the web-based program, OptiRNAi, to identify the most suitable regions of the target gene for RNAi design.