Systems NMR: Novel approach for studying biomolecular cell processes

The new approach, which was published on 29 July by Nature Methods, allows researchers to simultaneously, in one single sample at once, observe several proteins, metabolites and a nucleic acid (RNA), which represent three major classes of molecules of any cell. So far, one needed to use different methods to monitor different molecules. Reconstructing the overall picture of a network then required the “overlaying" of data recorded with different methods and samples. As an analogy: imagine that making everyday photos would require several different photo cameras to capture for example green-, red- and blue-colored objects. And each type of camera had a different resolution and focus depth. Overlaying snapshots from such different cameras might then obscure small details of the real scene. Not without limitations, the "Systems NMR" approach aims to address this challenge and to provide a holistic view on molecular networks. Monitoring multiple molecule and reaction types simultaneously in the same sample, the researcher may spot correlations between the behaviour of one molecule and the changes of another molecule, which could be missed if observed in different samples.

The second strong advantage of the new “Systems NMR” approach is that it allows not only to record snapshots of molecular mixtures but enables monitoring molecules in a dynamic fashion – similar to having a proper video instead of one or several photo snapshots. At the moment such continuous observations of network dynamics are only available in low-resolution methods, which observe only one or very few molecules at the same time. Researchers often have to choose between taking one high-resolution "photo" of a molecular network (e.g. with Mass-Spectrometry omics), or recording a low-resolution "video" (e.g. with biochemical assays) – without the ability to tell different network components apart. "Systems NMR" fills this gap allowing to record medium-resolution "videos" of molecular networks in which multiple molecular components can be easily distinguished at the same time, even with atom-level detail.
 

In fact, the atom-level resolution allows the new approach to combine both functional and structural information within the same NMR assay. The functions of molecules in the network are recorded in the form of speeds of reactions they take part in, and at the same time structural information is provided by the observed NMR signals with atom/residue-level resolution. In case of metabolites: at least individual hydrogen atoms can be distinguished; in case of proteins: individual amino acid residues in protein sequence; in case of structured RNAs: individual base-paired nucleotides.

The main current limitation of the approach is its relatively poor sensitivity - with conventional NMR currently requiring around micromolar concentrations for the target molecules to be observable.

Providing an experimental ground of intermediate complexity between simplified single-reaction assays and highly complex in vivo networks, the "Systems NMR" method can already reveal network-level behaviours not observed when monitoring only a few molecules. In one set of experiments, researchers placed five different RNA-binding proteins together in the presence of one RNA, and observed that the proteins are not just individually interacting with the target RNA, but are together forming some larger assembly which de-mixes (phase-separates) from the main solution – similar to oil separating from water. Such phase-separation phenomena are a new paradigm in cellular organization and are investigated in relation to various age-related disorders.

In view of potential applications, the method also allows to probe the effect of drug candidates simultaneously on different parts of a bio-molecular network. This may provide insights into the mechanism of drug action and can potentially reveal unintended (off-target) effects. Such analysis was done in the published paper, using drug candidates designed for the treatment of Spinal Muscular Atrophy, the leading genetic cause of infant mortality. The assays revealed that in addition to the expected interactions with the pre-existing RNA molecules, one of the tested molecules appeared to influence a different part of the network associated with the synthesis of RNA.

The new "Systems NMR" approach is expected to provide the strongest advance in the studies of so called heterotypic networks, which simultaneously involve different molecule classes and reaction types, and hence at the moment require a combination of several different methods to characterise them in detail. For example at points of cross-talk between cell signalling and metabolic networks, where perturbations of environment-sensing regulatory proteins are converted into changes of metabolites, which provide energy and building blocks for cell operation. A future combination of “Systems NMR” network quantification with “in-cell NMR” monitoring of molecules in living cells will deepen our understanding of network dynamics not just in vitro, but close to true physiological conditions.