Department of Biology, Institute of Molecular Biology & Biophysics, ETH Zurich, Zurich, Switzerland.
Department of Biology, Institute of Biochemistry, ETH Zurich, Zurich, Switzerland.
Nat Methods. 2019 Aug;16(8):743-749. doi: 10.1038/s41592-019-0495-7. Epub 2019 Jul 29.
Cellular behavior is controlled by the interplay of diverse biomolecules. Most experimental methods, however, can only monitor a single molecule class or reaction type at a time. We developed an in vitro nuclear magnetic resonance spectroscopy (NMR) approach, which permitted dynamic quantification of an entire 'heterotypic' network-simultaneously monitoring three distinct molecule classes (metabolites, proteins and RNA) and all elementary reaction types (bimolecular interactions, catalysis, unimolecular changes). Focusing on an eight-reaction co-transcriptional RNA folding network, in a single sample we recorded over 35 time points with over 170 observables each, and accurately determined five core reaction constants in multiplex. This reconstruction revealed unexpected cross-talk between the different reactions. We further observed dynamic phase-separation in a system of five distinct RNA-binding domains in the course of the RNA transcription reaction. Our Systems NMR approach provides a deeper understanding of biological network dynamics by combining the dynamic resolution of biochemical assays and the multiplexing ability of 'omics'.
细胞行为受多种生物分子相互作用的控制。然而，大多数实验方法一次只能监测单一分子类别或反应类型。我们开发了一种体外核磁共振光谱（NMR）方法，能够动态定量整个“异质”网络——同时监测三种不同的分子类别（代谢物、蛋白质和 RNA）和所有基本反应类型（双分子相互作用、催化、单分子变化）。我们聚焦于一个八反应共转录 RNA 折叠网络，在一个样本中记录了超过 35 个时间点，每个时间点有超过 170 个可观测变量，并在多路复用中准确确定了五个核心反应常数。这种重构揭示了不同反应之间意想不到的串扰。我们还在 RNA 转录反应过程中观察到五个不同 RNA 结合结构域的动态相分离。我们的系统 NMR 方法通过结合生化测定的动态分辨率和“组学”的多路复用能力，为深入了解生物网络动态提供了可能。
