OligoWalk: an online siRNA design tool utilizing hybridization thermodynamics

Abstract

Given an mRNA sequence as input, the OligoWalk web server generates a list of small interfering RNA (siRNA) candidate sequences, ranked by the probability of being efficient siRNA (silencing efficacy greater than 70%). To accomplish this, the server predicts the free energy changes of the hybridization of an siRNA to a target mRNA, considering both siRNA and mRNA self-structure. The free energy changes of the structures are rigorously calculated using a partition function calculation. By changing advanced options, the free energy changes can also be calculated using less rigorous lowest free energy structure or suboptimal structure prediction methods for the purpose of comparison. Considering the predicted free energy changes and local siRNA sequence features, the server selects efficient siRNA with high accuracy using a support vector machine. On average, the fraction of efficient siRNAs selected by the server that will be efficient at silencing is 78.6%. The OligoWalk web server is freely accessible through internet at http://rna.urmc.rochester.edu/servers/oligowalk.

A dynamic metabolite valve for the control of central carbon metabolism

“…While relatively uncommon in prokaryotic systems, there has been increasing interest in the use of RNAbased gene expression control systems for biotechnology applications (Biedendieck et al, 2009;Callura et al, 2010Callura et al, , 2012Desai and Papoutsakis, 1999;Isaacs et al, 2004;Kim et al, 2009;Kim and Cha, 2003;Tummala et al, 2003aTummala et al, , 2003b. The few examples of such natural and engineered systems extant in the literature are diverse in their approach with equally diverse design rules for asRNA constructs stressing the strength of the binding interaction (Lu and Mathews, 2008;Mutalik et al, 2012), kinetics of hybridization (Patzel and Sczakiel, 1998;Wang and Drlica, 2003) or the structure of the construct itself (Patzel and Sczakiel, 1998;Tummala et al, 2003b). Successful designs have been noted to be short (o100 nts) (Callura et al, 2010(Callura et al, , 2012Good, 2003;Isaacs et al, 2004) or long with significant complementarity to the ORF (Biedendieck et al, 2009;Desai and Papoutsakis, 1999;Kim and Cha, 2003;Tummala et al, 2003aTummala et al, ,2003b; with single hairpins (Callura et al, 2012;Good, 2003) or more complex architecture for stability (Patzel and Sczakiel, 1998;Tummala et al, 2003b).…”

A novel siRNA validation system for functional screening of effective RNAi targets in mammalian cells and development of a derivative lentivirus delivery system

“…This vector contains the following three cassettes: (1) a firefly luciferase reporter gene expression cassette followed by a multiple cloning site and SV40 poly(A) for the insertion of a target sequence, which results in a fusion transcript of the target gene and reporter gene, including two independent ORFs; (2) siRNA expression cassette containing a dual PoI III promoter driving in opposite directions (U6 and H1 promoters), which results in a 19-23 nt, double-stranded, small RNA fragment (siRNA). Basing on two kinds of novel standard siRNA design algorithms, namely OligoWalk and RNAxs (Lu and Mathews, 2008;Tafer et al, 2008), we selected three shRNAs targeting different regions within the same gene that had the fewest amounts of BLAST matches, that did not overlap a region of SNP, and that targeted different regions within the gene. Through calculating thermodynamic features of senseantisense hybridization and analyzing the statistical mechanics of the siRNA-target interaction, the OligoWalk web server is constructed to predict efficient siRNA candidates for a given mRNA sequence.…”

Recent Advances in RNA Interference Therapeutics for CNS Diseases

“…There are many online tools and guidelines that help to design siRNA sequences to a gene target of interest [27][28][29][30]. Newer online tools also incorporate the very important aspect of siRNA off-targeting [31][32][33].…”