A Startling Technique: Solution Phase NMR of Live Single Cells for Metabolic Studies.
Early in my career, as I was into organic synthesis, I lived and died pretty much by NMR, simple NMR, 1D NMR.
The analytical technique most important to me these days is mass spec, and recently, as my career approaches its end, I have become aware of the use of flow cytometry which now allows for the isolation of single cells, which has many obvious biomedical applications. I am watching closely the approach of mass spectroscopic analysis.
Scientists like (among others) Vicki Wysocki at the University of Ohio and Caroline Bertozzi at Stanford have greatly advanced the mass spectrometry of biomolecules, the former in such areas as protein/protein interactions and complexes, and the latter, often mentioned as a candidate for the Nobel Prize, in diverse areas such as the very difficult area represented by the proteomic sugar code.
Mass spec, one of the most powerful analytical techniques in the world, however is inherently a destructive technique. You cannot use it on living systems.
Among the many fascinating advances in NMR of which I've learned is the ability to use it in "PAT," Process Analytical Technology - I, and some of the old folks I was with at the time - were somewhat shocked to learn that modern signal processing technology allows for the use of NMR without perdeuterated solvents.
Apparently "that ain't nothing, baby!"
I haven't touched NMR, except in an extremely peripheral fashion, for more than 25 years. Of course many people, even outside of the sciences, are aware of "MRI" "Magnetic Resonance Imaging" (from which the word "nuclear" was stripped to address the fear and assertions of ignorance the word inspires) for the imaging of biological tissues, but today in an issue of Chemical Reviews I learned that people are currently working on the single cell, live, as in living, NMR.
The paper that blows my mind:
Radio Signals from Live Cells: The Coming of Age of In-Cell Solution NMR Enrico Luchinat, Matteo Cremonini, and Lucia Banci, Chemical Reviews 2022 122 (10), 9267-9306.
Some excerpts from the introductory paragraphs:
...NMR spectroscopy is the only one able to obtain information on the structure, the kinetics, and the thermodynamics of biological macromolecules at the atomic level, as it can observe them in native-like environments at physiological temperatures, and it can do so in a nondestructive manner. (6) Such a feature has always made NMR spectroscopy appealing for the study of small and large molecules not only in vitro, isolated from their physiological context, but directly inside intact living cells. Compared to other spectroscopic techniques, NMR suffers from an intrinsically low sensitivity; therefore, its applicability to cells was traditionally restricted to the observation of small, highly abundant molecules. Indeed, in the past century, cellular NMR studies were mostly focused on the analysis of cellular metabolism, for example, by exploiting the observation of phosphorus-containing molecules through 31P NMR, or by introducing 13C-labeled precursors for a metabolic flux analysis. In some cases, very abundant small macromolecules could be studied, often because of peculiar properties that made them stand out against the rest of the milieu, as it is the case for highly shifted signals of paramagnetic metalloproteins. Then, in the early 2000s, it became clear that modern NMR spectrometers, with a higher magnetic field and more sensitive hardware, could detect signals from isotopically labeled proteins inside the bacteria in which they were recombinantly expressed. (7) Shortly after, macromolecules─proteins at first, then nucleic acids─were delivered to eukaryotic cells. The cellular NMR approach, reborn as “in-cell NMR”, soon gained widespread recognition, in a time when the scientific community had realized the importance of performing biochemical and biophysical studies in physiologically relevant contexts, and huge advancements were being made in developing techniques, such as single-molecule Förster resonance energy transfer (FRET) and cryo-electron tomography, that would be able to characterize macromolecules in a cellular environment...
...This work provides a detailed overview of the development and applications of in-cell solution NMR approaches during the first ∼20 years since its inception in the modern sense. We first describe the existing approaches for cell sample preparation, the various types and strategies for isotopic incorporation, and the NMR methods that can be applied to living cells. We then review the application of in-cell NMR to different biological questions: how the cellular environment affects the folding thermodynamics of a protein, its structural and dynamic properties, and its interactions with specific cellular partners; whether the structure of a folded protein in cells differs from that determined in vitro...
This is unbelievable.
It's been going on for 20 years, and I missed it entirely.
At the end of my life, I feel great anxiety and shame about what my generation is leaving for future generations: They face unbelievable risks as a result of our unrealized fantasies. A saving grace, perhaps, for all the evil we have done, is that we have left for them a record of unbelievably high technology, the tools from which perhaps, they can escape the miasma of shame we have left for them.
This may be esoteric, but the implications are enormous.
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