title: 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 (Km=74 nM) with substrate inhibition kinetics (Ki=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.
authors: Department of Target Biology, Pfizer Oligonucleotide Therapeutics Unit, Cambridge South Campus, Cambridge, Massachusetts, United States of America; Department of Protein Biology, Pfizer Global Biotherapeutics Technology Unit, Cambridge North Campus, Cambridge, Massachusetts, United States of America; Department of Medicine, Division of Pulmonary, Critical Care and Sleep Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, United States of America.
authors: Mark A
authors: MA
authors: Bernard
authors: Department of Target Biology, Pfizer Oligonucleotide Therapeutics Unit, Cambridge South Campus, Cambridge, Massachusetts, United States of America.
authors: Leyu
authors: L
authors: Wang
authors: Department of Medicine, Division of Pulmonary, Critical Care and Sleep Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, United States of America.
authors: Souvenir D
authors: SD
authors: Tachado
mesh_terms: D060565:Argonaute Proteins / Q000378:metabolism*; D001667:Binding, Competitive; D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics; D007700:Kinetics; D011485:Protein Binding; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000378:metabolism*; D043244:Ribonuclease III / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics; D013940:Thymidylate Synthase / Q000378:metabolism
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D013940:Thymidylate Synthase; D043244:Ribonuclease III