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Published online before print March 26, 2008, 10.1261/rna.704708
RNA (2008), 14:853-861. Published by Cold Spring Harbor Laboratory Press. Copyright © 2008 RNA Society.
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Experimental validation of the importance of seed complement frequency to siRNA specificity

Emily M. Anderson, Amanda Birmingham, Scott Baskerville, Angela Reynolds, Elena Maksimova, Devin Leake, Yuriy Fedorov, Jon Karpilow, and Anastasia Khvorova1

Thermo Fisher Scientific, Dharmacon Products, Lafayette, Colorado 80026, USA

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: seed complement frequencies (SCFs); RNA interference (RNAi); small interfering RNAs (siRNA); microRNAs (miRNAs); RNA induced silencing complex (RISC)

Received June 27, 2007 ; accepted October 24, 2007.


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