title: Terminal Duplex Stability and Nucleotide Identity Differentially Control siRNA Loading and Activity in RNA Interference.
abstract: Efficient short interfering RNA (siRNA)-mediated gene silencing requires selection of a sequence that is complementary to the intended target and possesses sequence and structural features that encourage favorable functional interactions with the RNA interference (RNAi) pathway proteins. In this study, we investigated how terminal sequence and structural characteristics of siRNAs contribute to siRNA strand loading and silencing activity and how these characteristics ultimately result in a functionally asymmetric duplex in cultured HeLa cells. Our results reiterate that the most important characteristic in determining siRNA activity is the 5' terminal nucleotide identity. Our findings further suggest that siRNA loading is controlled principally by the hybridization stability of the 5' terminus (Nucleotides: 1-2) of each siRNA strand, independent of the opposing terminus. Postloading, RNA-induced silencing complex (RISC)-specific activity was found to be improved by lower hybridization stability in the 5' terminus (Nucleotides: 3-4) of the loaded siRNA strand and greater hybridization stability toward the 3' terminus (Nucleotides: 17-18). Concomitantly, specific recognition of the 5' terminal nucleotide sequence by human Argonaute 2 (Ago2) improves RISC half-life. These findings indicate that careful selection of siRNA sequences can maximize both the loading and the specific activity of the intended guide strand.
authors.affiliation: Department of Chemical Engineering and Materials Science, Michigan State University , East Lansing, Michigan.
authors.forename: Phillip A
authors.initials: PA
authors.lastname: Angart
authors.affiliation: Department of Chemical Engineering and Materials Science, Michigan State University , East Lansing, Michigan.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Carlson
authors.affiliation: Department of Chemical Engineering and Materials Science, Michigan State University , East Lansing, Michigan.
authors.forename: Kwasi
authors.initials: K
authors.lastname: Adu-Berchie
authors.affiliation: Department of Chemical Engineering and Materials Science, Michigan State University , East Lansing, Michigan.
authors.forename: S Patrick
authors.initials: SP
authors.lastname: Walton
mesh_terms: D060565:Argonaute Proteins / Q000235:genetics* / Q000378:metabolism; D006207:Half-Life; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D034622:RNA Interference; D020871:RNA Stability; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex
keywords: Ago2; RNAi; duplex thermodynamics; functional asymmetry; siRNA; terminal nucleotide