Experimental validation of the importance of seed complement frequency to siRNA specificity.
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.
Anderson
Emily M
EM
Thermo Fisher Scientific, Dharmacon Products, Lafayette, Colorado 80026, USA.
Birmingham
Amanda
A
Baskerville
Scott
S
Reynolds
Angela
A
Maksimova
Elena
E
Leake
Devin
D
Fedorov
Yuriy
Y
Karpilow
Jon
J
Khvorova
Anastasia
A
D020413:3' Untranslated Regions; D001483:Base Sequence; D020869:Gene Expression Profiling; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D020407:Internet; D008957:Models, Genetic; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000378:metabolism; D014162:Transfection
D020413:3' Untranslated Regions; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex