Abstract

Pairing between the hexamer seed region of a small interfering RNA (siRNA) guide strand (nucleotides 2–7) and complementary sequences in the 3′ UTR of mature transcripts has been implicated as an important element in off-target gene regulation and false positive phenotypes. To better understand the association between seed sequences and off-target profiles we performed an analysis of all possible (4096) hexamers and identified a nonuniform distribution of hexamer frequencies across the 3′ UTR transcriptome. Subsequent microarray analysis of cells transfected with siRNAs having seeds with low, medium, or high seed complement frequencies (SCFs) revealed that duplexes with low SCFs generally induced fewer off-targets and off-target phenotypes than molecules with more abundant 3′ UTR complements. These findings provide the first experimentally validated strategy for designing siRNAs with enhanced specificity and allow for more accurate interpretation of high throughput screening data generated with existing siRNA/shRNA collections.

Keywords

BiologyRandom hexamerSmall interfering RNARNA interferenceComputational biologyTranscriptomeUntranslated regionGeneticsPhenotypeRNAGeneCell biologyMolecular biologyGene expression

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Year
2008
Type
article
Volume
14
Issue
5
Pages
853-861
Citations
144
Access
Closed

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Emily M. Anderson, Amanda Birmingham, Scott Baskerville et al. (2008). Experimental validation of the importance of seed complement frequency to siRNA specificity. RNA , 14 (5) , 853-861. https://doi.org/10.1261/rna.704708

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DOI
10.1261/rna.704708