Terminal Duplex Stability and Nucleotide Identity Differentially Control siRNA Loading and Activity in RNA Interference.
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.
['Angart|Phillip A|PA|', 'Carlson|Rebecca J|RJ|', 'Adu-Berchie|Kwasi|K|', 'Walton|S Patrick|SP|']
D060565:Argonaute Proteins D006207:Half-Life D006367:HeLa Cells D006801:Humans D007700:Kinetics D009693:Nucleic Acid Hybridization D034622:RNA Interference D020871:RNA Stability D012333:RNA, Messenger D034741:RNA, Small Interfering D034743:RNA-Induced Silencing Complex D013329:Structure-Activity Relationship D013816:Thermodynamics
siRNA RNAi terminal nucleotide Ago2 duplex thermodynamics functional asymmetry