Systems NMR: single-sample quantification of RNA, proteins, and metabolites for biomolecular network analysis

Cellular behavior is controlled by the interplay of diverse biomolecules. Most experimental methods, however, can monitor only 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, RNA) and all elementary reaction types (bimolecular interactions, catalysis, unimolecular changes). Focusing on an 8-reaction co-transcriptional RNA folding network, in a single sample we recorded over 35 time-points with over 170 observables each, and accurately determined 5 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”.