Dissecting RNA-Interference Pathway with Small Molecules

Abstract

RNA interference (RNAi) is a process whereby short-interfering RNAs (siRNA) silence gene expression in a sequence-specific manner. We have screened a chemical library of substituted dihydropteridinones and identified a nontoxic, cell permeable, and reversible inhibitor of the RNAi pathway in human cells. Biochemical and fluorescence resonance-energy transfer experiments demonstrated that one of the compounds, named ATPA-18, inhibited siRNA unwinding that occurred within 6 hr of siRNA transfection. Extracts prepared from ATPA-18-treated cells also exhibited a decrease in target RNA cleavage by activated RNA-induced silencing complex (RISC*). Interestingly, when activated RISC*, which harbors unwound antisense siRNA, was treated with ATPA-18 in vitro, target RNA cleavage was not affected, indicating that this compound inhibited siRNA unwinding or steps upstream of unwinding in the RNAi pathway. Our results also establish the timing of siRNA unwinding and show that siRNA helicase activity is required for RNAi. ATPA-18 analogs will therefore provide a new class of small molecules for studying RNAi mechanisms in a variety of model organisms and deciphering in vivo genetic functions through reverse genetics.

Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA

“…As shown by hybridization studies, duplexes aG2/as2Uall 19 bp (19), aG2/as2Uall 17 bp (20), and aG2/ as2Uall 15 bp (21) exhibit extremely high thermodynamic stability (the relevant melting profiles did not reach a plateau up to 96°C). We asked the question whether such stable duplexes can be effectively unwound by the dsRNA helicase engaged in the RISC complex (Chiu et al 2005). It has already been reported that thermodynamically stable duplexes containing LNA-modified units are active in RNAi-induced gene silencing (Braasch et al 2003;Elmen et al 2005), although their ethylene bridged nucleic acid analogs completely lost their silencing potency (Hamada et al 2002).…”

Artificial control of gene expression in mammalian cells by modulating RNA interference through aptamer–small molecule interaction

“…To quantitatively determine in vivo RNAi activity, dual fluorescence reporter systems using EGFP and DsRed (RFP) have been developed and used by many researchers Rana 2002, 2003;Chiu et al 2004Chiu et al , 2005Shah et al 2005;Yang et al 2005). Our assay procedure was established according to the previous reports with some modifications.…”

Chemical Biology Approach for Dissection of RNAi/miRNA Pathway

“…The criterion applied for evaluation of the drugmediated siRNA effects was the ratio of EGFP/RFP fluorescence in the presence of siRNA calculated and normalized to the EGFP/RFP ratio of mock treated cells. Using this assay strategy, two compounds named ATPA-18 and ATPA-21 were identified to specifically inhibit ATP-dependent events occurring during RNAi [31]. A series of in vitro and in vivo analyses demonstrated that these compounds specifically affected an early unwinding step in the RNAi pathway, suggesting that its target is an ATP-dependent RNA helicase, although the precise molecular target remains to be elusive [31].…”

“…Using this assay strategy, two compounds named ATPA-18 and ATPA-21 were identified to specifically inhibit ATP-dependent events occurring during RNAi [31]. A series of in vitro and in vivo analyses demonstrated that these compounds specifically affected an early unwinding step in the RNAi pathway, suggesting that its target is an ATP-dependent RNA helicase, although the precise molecular target remains to be elusive [31]. While these compounds were the first inhibitors for the siRNA activity, they had no effect on the endogenous miRNA pathway, establishing the timing of siRNAs' unwinding and suggesting that siRNA helicase activity is required for RNAi.…”

“…In vivo fluorescence-based reporter system was initially developed by transient co-transfection of plasmids harboring enhanced green-and red-fluorescent proteins (EGFP and RFP, respectively) into HeLa cells together with siEGFP against its targets EGFP mRNA for degradation [31]. The co-transfected Hela cells were treated with a synthetic small chemical library.…”