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

Abstract

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 cotranscriptional 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".Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Hydrolysis of Extracellular ATP by Vascular Smooth Muscle Cells Transdifferentiated into Chondrocytes Generates Pi but Not PPi

“…Thanks to the natural abundance of phosphorous, 31 P-NMR is a convenient method to quantify various nucleotides in a mixture (e.g., ATP hydrolysis products) without isolation (e.g., chromatographic analysis) or/and labeling [ 38 ]. Additionally, it does not require the use of radioactive species.…”

“…Additionally, it does not require the use of radioactive species. Moreover, as a non-destructive method, it is particularly adapted to follow reaction kinetics [ 38 ]. Here, extracellular reaction media were extracted periodically from MOVAS to record 31 P-NMR spectra from the whole reaction system, avoiding interference from intracellular nucleotides, nucleic acids, and phospholipids.…”

“…31 P-NMR spectra were measured with a Ascend™ 600 (Bruker ® , Birrica, MA, USA) or with a 300 ultrashield (Bruker ® ) spectrometer. Simultaneous ATP, ADP, AMP, PP i , and P i peaks were monitored [ 38 ]. Peaks were integrated to determine the concentrations of species based on the total phosphorous content (150 mM for), thanks to the natural abundancy of 31 P. At least three independent measurements were performed to obtain kinetics parameters.…”

Zirconia Hybrid Nanoshells for Nutrient and Toxin Detection

“…Various detection methods to detect residues of melamine in dairy products were summarized in Table S5, Supporting Information, including liquid chromatography, [ 39 ] ELISA, [ 40 ] nuclear magnetic resonance (NMR) spectroscopy, [ 41 ] and biochemical sensing [ 42 ] etc. However, the common detection techniques are time‐consuming (e.g., ≈40 min for chromatographic separation), [ 43 ] and labor‐intensive (e.g., cleanup procedures required in NMR) [ 44 ] with short shelf life (e.g., <1 year for biochemical sensors), [ 42,45 ] far from ideal for high‐throughput sample screening. In comparison, the zirconia nanoshell‐assisted LDI MS established a direct (no sample pretreatment) and fast (≈1 min for one sample) method for the sensitive (≈p m ) and selective detection of melamine in dairy products, which could be applied to real‐time and on‐site analysis.…”

“…timeconsuming (e.g., ≈40 min for chromatographic separa tion), [43] and laborintensive (e.g., cleanup procedures required in NMR) [44] with short shelf life (e.g., <1 year for biochemical sensors), [42,45] far from ideal for highthroughput sample screening. In comparison, the zirconia nanoshellassisted LDI MS established a direct (no sample pretreatment) and fast (≈1 min for one sample) method for the sensitive (≈pm) and selective detection of melamine in dairy products, which could be applied to realtime and onsite analysis.…”