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Despite the promise of short interfering RNAs (siRNA), contending with off-target is a challenge for RNAi users. To alleviate these problems, we have developed locked nucleic acid (LNA) modified siRNAs and optimized performance using cellular phenotypic assays as well as microarray analysis. During development, we compared LNA and 2'OMethoxy (2'OMe) chemistries placed strategically throughout the siRNA molecule and found a novel pattern of LNA placement that greatly improved the specificity of the siRNA and reduced it's toxicity in culture while preserving the potency of the siRNA. The improvements in specificity made by LNA-modified siRNAs were developed and validated by measuring the phenotypic signatures in a high content cell-based screening assay as well as comparison of the level of differentially expressed genes observed in microarray analysis between modified and unmodified siRNAs. HT screening of a collection of genes demonstrated that the LNA-modified siRNAs exhibits the best overall rate to elicit the expected phenotype, reduced toxicity and achieved an improved coherence of phenotype compared to 2'OMe-modified or unmodified siRNAs.

LNA(R) incorporated siRNAs exhibit lower off-target effects compared to 2'-OMethoxy in Cell Phenotypic Assays and Microarray Analysis