Bernard Mark A, Wang Leyu, Tachado Souvenir D
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.
Department of Target Biology, Pfizer Oligonucleotide Therapeutics Unit, Cambridge South Campus, Cambridge, Massachusetts, United States of America.
PLoS One. 2015 Mar 20;10(3):e0120614. doi: 10.1371/journal.pone.0120614. eCollection 2015.
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.
RNA 诱导沉默复合体（RISC）中RNA 加工的机制研究因缺乏连续监测酶活性的方法而受到阻碍。RNAi 酶的“无淬灭剂”荧光底物能够连续监测酶促反应，以进行详细的动力学研究。重组 RISC 酶可切割靶向人胸苷酸合成酶（TYMS）和缺氧诱导因子 1-α 亚基（HIF1A）的荧光底物。使用荧光双链 RNA DICER 底物和荧光 siRNA，DICER 与 Argonaute2 的混合物相对于单独的任何一种酶都表现出协同酶活性，并且添加 TRBP 不会增强表观活性。酶分析中对 AGO2 和 DICER 的滴定表明，AGO2 和 DICER 以等摩尔比形成功能性高亲和力复合物。DICER 和 DICER + AGO2 对 DICER 底物表现出米氏动力学。然而，没有 DICER 酶时，AGO2 无法加工荧光 siRNA，这表明 AGO2 无法将 siRNA 自加载到其活性位点。DICER + AGO2 组合以底物抑制动力学（Ki = 105 nM）加工荧光 siRNA 底物（Km = 74 nM），通过实验证明 siRNA 结合两个不同的位点，这些位点影响 RISC 中的切割和 AGO2 加载反应。该结果表明，结合在 DICER 活性位点的 siRNA（DICER 的产物）可能直接转移（作为 AGO2 底物）到 DICER + AGO2 复合物中 AGO2 的活性位点。竞争性底物分析表明，DICER + AGO2 对荧光 siRNA 的切割具有特异性，因为未标记的 siRNA 和 DICER 底物作为竞争性底物，会导致荧光速率呈浓度依赖性下降。一系列 DICER 底物的体外竞争性底物分析与基于细胞的 HIF1A mRNA 敲低分析相关（对数-对数斜率 = 0.29），这表明改进的 DICER 底物设计可使 DICER + AGO2 复合物的加工能力提高 10 倍，从而可使 mRNA 敲低效力大幅提高（约 2800 倍）。本研究为优化基于核酸的治疗方法奠定了基础，该方法用于切割和 Argonaute2 加载以提高疗效，采用了系统的生化方法。
