siDirect (http://design.RNAi.jp/) is a web-based online software system for computing highly effective small interfering RNA (siRNA) sequences with maximum target-specificity for mammalian RNA interference (RNAi). Highly effective siRNA sequences are selected using novel guidelines that were established through an extensive study of the relationship between siRNA sequences and RNAi activity. Our efficient software avoids off-target gene silencing to enumerate potential cross-hybridization candidates that the widely used BLAST search may overlook. The website accepts an arbitrary sequence as input and quickly returns siRNA candidates, providing a wide scope of applications in mammalian RNAi, including systematic functional genomics and therapeutic gene silencing.
Designing highly effective short interfering RNA (siRNA) sequences with maximum target-specificity for mammalian RNA interference (RNAi) is one of the hottest topics in molecular biology. The relationship between siRNA sequences and RNAi activity has been studied extensively to establish rules for selecting highly effective sequences. However, there is a pressing need to compute siRNA sequences that minimize off-target silencing effects efficiently and to match any non-targeted sequences with mismatches.
The enumeration of potential cross-hybridization candidates is non-trivial, because siRNA sequences are short, ca. 19 nt in length, and at least three mismatches with non-targets are required. With at least three mismatches, there are typically four or five contiguous matches, so that a BLAST search frequently overlooks off-target candidates. By contrast, existing accurate approaches are expensive to execute; thus we need to develop an accurate, efficient algorithm that uses seed hashing, the pigeonhole principle, and combinatorics to identify mismatch patterns. Tests show that our method can list potential cross-hybridization candidates for any siRNA sequence of selected human gene rapidly, outperforming traditional methods by orders of magnitude in terms of computational performance.
RNA interference (RNAi), mediated by 21-nucleotide (nt)-length small interfering RNAs (siRNAs), is a powerful tool not only for studying gene function but also for therapeutic applications. RNAi, requiring perfect complementarity between the siRNA guide strand and the target mRNA, was believed to be extremely specific. However, a recent growing body of evidence has suggested that siRNA could down-regulate unintended genes whose transcripts possess complementarity to the 7-nt siRNA seed region. This off-target gene silencing may often provide incongruous results obtained from knockdown experiments, leading to misinterpretation. Thus, an efficient algorithm for designing functional siRNAs with minimal off-target effect based on the mechanistic features is considered of value.
We present siDirect 2.0, an update of our web-based software siDirect, which provides functional and off-target minimized siRNA design for mammalian RNAi. The previous version of our software designed functional siRNAs by considering the relationship between siRNA sequence and RNAi activity, and provided them along with the enumeration of potential off-target gene candidates by using a fast and sensitive homology search algorithm. In the new version, the siRNA design algorithm is extensively updated to eliminate off-target effects by reflecting our recent finding that the capability of siRNA to induce off-target effect is highly correlated to the thermodynamic stability, or the melting temperature (Tm), of the seed-target duplex, which is formed between the nucleotides positioned at 2-8 from the 5' end of the siRNA guide strand and its target mRNA. Selection of siRNAs with lower seed-target duplex stabilities (benchmark Tm < 21.5 degrees C) followed by the elimination of unrelated transcripts with nearly perfect match should minimize the off-target effects.
siDirect 2.0 provides functional, target-specific siRNA design with the updated algorithm which significantly reduces off-target silencing. When the candidate functional siRNAs could form seed-target duplexes with Tm values below 21.5 degrees C, and their 19-nt regions spanning positions 2-20 of both strands have at least two mismatches to any other non-targeted transcripts, siDirect 2.0 can design at least one qualified siRNA for >94% of human mRNA sequences in RefSeq. siDirect 2.0 is available at http://siDirect2.RNAi.jp/.
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