DICER-ARGONAUTE2 Complex in Continuous Fluorogenic Assays of RNA Interference Enzymes

Abstract

Mechanistic studies of RNA processing in the RNA-Induced Silencing Complex (RISC) have been hindered by lack of methods for continuous monitoring of enzymatic activity. “Quencherless” fluorogenic substrates of RNAi enzymes enable continuous monitoring of enzymatic reactions for detailed kinetics studies. Recombinant RISC enzymes cleave the fluorogenic substrates targeting human thymidylate synthase (TYMS) and hypoxia-inducible factor 1-α subunit (HIF1A). Using fluorogenic dsRNA DICER substrates and fluorogenic siRNA, DICER+ARGONAUTE2 mixtures exhibit synergistic enzymatic activity relative to either enzyme alone, and addition of TRBP does not enhance the apparent activity. Titration of AGO2 and DICER in enzyme assays suggests that AGO2 and DICER form a functional high-affinity complex in equimolar ratio. DICER and DICER+AGO2 exhibit Michaelis-Menten kinetics with DICER substrates. However, AGO2 cannot process the fluorogenic siRNA without DICER enzyme, suggesting that AGO2 cannot self-load siRNA into its active site. The DICER+AGO2 combination processes the fluorogenic siRNA substrate (K m=74 nM) with substrate inhibition kinetics (K i=105 nM), demonstrating experimentally that siRNA binds two different sites that affect Dicing and AGO2-loading reactions in RISC. This result suggests that siRNA (product of DICER) bound in the active site of DICER may undergo direct transfer (as AGO2 substrate) to the active site of AGO2 in the DICER+AGO2 complex. Competitive substrate assays indicate that DICER+AGO2 cleavage of fluorogenic siRNA is specific, since unlabeled siRNA and DICER substrates serve as competing substrates that cause a concentration-dependent decrease in fluorescent rates. Competitive substrate assays of a series of DICER substrates in vitro were correlated with cell-based assays of HIF1A mRNA knockdown (log-log slope=0.29), suggesting that improved DICER substrate designs with 10-fold greater processing by the DICER+AGO2 complex can provide a strong (~2800-fold) improvement in potency for mRNA knockdown. This study lays the foundation of a systematic biochemical approach to optimize nucleic acid-based therapeutics for Dicing and ARGONAUTE2-loading for improving efficacy.

Terminal Duplex Stability and Nucleotide Identity Differentially Control siRNA Loading and Activity in RNA Interference

“…After delivery of the siRNA to the cell cytoplasm, efficient guide strand loading requires nucleotide-independent phosphorylation of the siRNA 5¢ TNs [35], outcompeting native miRNAs for binding to Ago2 [36,37], conformational changes within Ago2 facilitated by HSP90 and potentially other proteins [10,[38][39][40][41], and cleavage and removal of the passenger strand [42,43]. We observed a large variation in the total amount of Ago2 loading even among siRNA strands with the same TN ( Supplementary Fig.…”

Analysis of Argonaute 2–microRNA complexes in ex vivo stored red blood cells

“…22 nucleotides) in 24 hours. In other mammalian cells, such RNA–protein complexes have been shown to be the functional unit of a RISC that is recruited on to a specific mRNA and regulate translation . Through functional enrichment analysis of mRNA targets of the 28 most abundant miRNAs bound to AGO2 we have identified targets related to various diseases (cancer), biofunctions, and toxicity functions such as cardiotoxicity and toxicities of specific organs involved (liver and kidneys) in RBC clearance and production (Table S1).…”

Computational search of hybrid human/SARS-CoV-2 dsRNA reveals unique viral sequences that diverge from those of other coronavirus strains

“…Dicer can trap dsRNAs pairing over a 19-base length with at least a 2 nt overhang at the 3 0 ends and can then directly transfer the modified/ unmodified siRNA to the Ago2 site (Snead and Rossi, 2012;Snead et al, 2013;Sun et al, 2008). This mechanism has been further demonstrated with fluorogenic dsRNA (Bernard et al, 2015). Some paired 19-mers with a dTdT overhang at the 3 0 end bind Dicer with high affinity; these sequences are not cleaved by Dicer, are fully transferred to Ago and trigger high-efficiency gene-silencing activity (Snead and Rossi, 2012;Snead et al, 2013).…”