Small molecule inhibition of RISC loading.
Argonaute proteins are the core components of the microRNP/RISC. The biogenesis and function of microRNAs and endo- and exo- siRNAs are regulated by Ago2, an Argonaute protein with RNA binding and nuclease activities. Currently, there are no in vitro assays suitable for large-scale screening of microRNP/RISC loading modulators. We describe a novel in vitro assay that is based on fluorescence polarization of TAMRA-labeled RNAs loaded to human Ago2. Using this assay, we identified potent small-molecule inhibitors of RISC loading, including aurintricarboxylic acid (IC(50) = 0.47 μM), suramin (IC(50) = 0.69 μM), and oxidopamine HCL (IC(50) = 1.61 μM). Small molecules identified by this biochemical screening assay also inhibited siRNA loading to endogenous Ago2 in cultured cells.
Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
Grace S
GS
Tan
Chun-Hao
CH
Chiu
Barry G
BG
Garchow
David
D
Metzler
Scott L
SL
Diamond
Marianthi
M
Kiriakidou
D060565:Argonaute Proteins / Q000378:metabolism*; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D012313:RNA / Q000032:analysis* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000037:antagonists & inhibitors / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012235:Rhodamines / Q000032:analysis; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000494:pharmacology*
C114557:5-carboxytetramethylrhodamine succinimidyl ester; C466422:AGO2 protein, human; D060565:Argonaute Proteins; D005456:Fluorescent Dyes; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D012235:Rhodamines; D054852:Small Molecule Libraries; D012313:RNA; D004247:DNA