Abstract
RNA interference (RNAi) has been widely applied in genome-scale gene functional screens. Traditional one-on-one RNAi analysis works well but is cost-ineffective and laborious. Previous studies have indicated that siRNAs actually can simultaneously affect RNAs that are near-perfectly complementary to the induced siRNAs, and this phenomenon has been termed as off-target effect. Off-target effect implies that it is possible to silence several genes simultaneously with a carefully designed siRNA. We first proposed a heuristic algorithm to design suitable siRNAs that can target multiple genes, and then adopted group testing method to present a gene functional analysis strategy. Combining these two work can help to reduce the number of required RNAi experiments in a large-scale RNAi analysis. To verify the efficacy of our strategy, we used the Orchid expressed sequence tag data as a case study to screen the putative transcription factors that are involved in plant disease responses. According to our computation, 94 qualified siRNAs were sufficient to examine all of the predicated 229 transcription factors. In addition, among the 94 computer-designed siRNAs, an siRNA that targets both TF15 (a previously identified transcription factor that is involved in the plant disease-response pathway) and TF21 was introduced into orchids. The experimental results showed that this siRNA can simultaneously silence TF15 and TF21, and application of our strategy successfully confirmed that TF15 is involved in plant defense responses. Interestingly, our second-round analysis, which used an siRNA specific to TF21, indicated that TF21 is a previously unidentified transcription factor that is related to plant defense responses. Our computational results showed that it is possible to screen all genes with fewer experiments than would be required for the traditional one-on-one RNAi screening. We also verified that our strategy is capable of identifying genes that are involved in a specific phenotype.