Israeli scientists collaborate to speed up Covid-19 RNA research using ultra-sensitive NMR techniques

Professor Lucio Frydman has a remarkable level of hands-on experience in nuclear magnetic resonance (NMR), having spent the first 15 years of his career using instruments that he built himself. His passion for the technology has remained undimmed – even after three-plus decades in the field. As he says, “I’m always excited to see what each day will bring,” and the last year brought many new discoveries for Prof. Frydman and his team, thanks to their involvement in a research consortium looking at the RNA structure of SARS-CoV-2, the virus causing Covid-19.

The shared language of NMR
The Weizmann Institute of Science in Rehovot, Israel is home to 250 research groups spanning all disciplines – from biology, biochemistry and chemistry, to physics, mathematics and computer science. The institute has always been one of the first to take advantage of new advances in instrumentation. These include (at various times) a superconducting 270 MHz NMR instrument, an 800 MHz system, a 7 T human MRI scanner, a 15.2 T animal MRI scanner, and – of greatest relevance to Prof. Frydman’s RNA work – a 1.0 GHz Bruker instrument.
Prof. Frydman describes his work as encompassing two complementary fields. The first of these is the study of NMR as a subject in its own right – for example, how nuclear spins behave in particular circumstances, and how to improve analytical sensitivity, resolution, and speed. The other aspect is the application of this knowledge, with Prof. Frydman’s team working in areas as diverse as materials, pharmaceuticals, RNA, and cancer.[1]
However, as Prof. Frydman points out, whatever the application, all NMR and magnetic resonance imaging (MRI) experiments are based on the same fundamental physical process – the clever disturbance of the equilibrium that nuclear and electron spins will achieve when placed in a strong magnetic field, by radio-frequency pulses. This “shared language”, as Prof. Frydman describes it, opens special avenues for cross-fertilization of ideas. For example, he says: “You can take an idea developed for imaging and apply it to a chemical context ... or a method developed for solid-state work and apply it to molecules in cells.”

Collaborating on Covid-19
It’s exactly this concept of ‘cross-fertilization’ that lies at the heart of Prof. Frydman’s most recent work, as part of the Covid-19 NMR Project.[2] This consortium of over 50 research groups from across the world aims to determine the structure of SARS-CoV-2’s RNA and if its proteins using NMR spectroscopy and, in doing so, to provide insights into their ‘drugability’ by small molecules.

Prof. Frydman’s own involvement in the Covid-19 NMR consortium arose from his previous connections to the Goethe-Universität in Frankfurt, Germany. The work had mostly involved improving the sensitivity and speed of RNA analysis, making him a natural collaborator when Professor Dr. Harald Schwalbe at the Goethe-Universität set up the consortium in March 2020. After joining this team, Prof. Frydman quickly shifted his efforts to studying RNA fragments from the large genome of SARS-CoV-2, using the 1 GHz Bruker, which had been installed at the Weizmann Institute in 2018, as well as other instruments.

The work is inherently collaborative, which Prof. Frydman says makes the experience a very positive one: “We’ve shared samples, methods, data libraries, results and more... all of which has helped us develop user-friendly methods for extracting data at high sensitivity more quickly.”

All this high-sensitivity data has resulted in a better understanding of the SARS-CoV-2 genome, including the characterization of the primary and secondary structure of numerous parts of the RNA structure,[3] and the discovery of several potential drugs that could bind to the RNA target. For his own part, Prof. Frydman highlights the benefits of having such a challenging task: “Being presented with the difficulties posed by Covid-19 has focused attention on the performance of the tools we have available. As well as helping us to understand the RNA structure of SARS-CoV-2, it’s very likely that these tools will ultimately find application in other fields – such is the nature of NMR.”

Exquisite detail with highly sensitive NMR techniques
One such tool that Prof. Frydman is particularly enthusiastic about is nuclear hyperpolarization [4, 5]. Briefly, this involves transferring the high degree of polarization of electron spins in a population of radicals to the nuclear spins of either water, or of 13C-labelled small molecules. This is carried out at low temperature, high magnetic field, and under microwave irradiation. The result is that the nuclei become polarized to a degree several orders of magnitude greater than normal, which in turn means that they can be studied at the very low concentrations needed for both in vitro and in vivo experiments.

Prof. Frydman highlights this as one example of the unexpected way in which scientific discoveries can lead to application benefits: “Nuclear hyperpolarization had been known about for 60 years, and for the first 40 of those years it was to a large extent a curiosity. But now it has found several concrete applications – just one of many examples in the field, where very basic knowledge on the behavior of elementary particles ultimately leads to tangible insights into learning about biomolecules and metabolism.”

Another technique that has found application in the Covid-19 work by the group concerns a family of magnetization transfer experiments [6, 7, 8] which overcome the low sensitivity and poor peak resolution encountered when attempting to determine the proximity of amino or imino protons to neighbouring protons using two-dimensional nuclear Overhauser effect spectroscopy (NOESY) and related techniques. The resolution of this approach, particularly at high fields, is comparable to that of conventional acquisitions, but its sensitivity is considerably enhanced. Magnetization transfer-based methods also enable experiments to be run at higher temperatures, opening the prospect of investigating RNA structures under physiological conditions.

Better tools, better understanding
“The techniques described above are just two examples of new technical advances driving better outcomes for society,” says Prof. Frydman, adding: “There’s a ‘pyramid of needs’ in this area of science – better tools result in better sensitivity and improved speed, enabling the study of more diverse samples under more realistic conditions, which in turn leads to a better understanding of natural systems.”

And what of the future? So far, Prof. Frydman’s work within the consortium has focused on obtaining a basic understanding of the RNA genome, performed by cutting the RNA into smaller fragments and working on them independently. “The challenge is now to understand the mechanism of action of proposed drugs, and to study larger, more dynamic RNA fragments. In particular, we want to see whether their structure, activity, and drug-binding patterns are preserved, especially at higher temperatures”, Prof. Frydman says.

Expert technical support from Bruker
Both prior to and during the Covid-19 project, the contribution from Bruker has been essential, says Prof. Frydman. “The Weizmann Institute has been a Bruker customer since the 1970s, but I first started using their instruments about eight years ago. My experience has been very positive, and I’m very grateful for everything they’ve done. Right from the start, they gave me a lot of help with integrating workflows for my older machines with new Bruker instruments, and in the years since then they’ve continued to provide expert technical support.”
Prof. Frydman continues that, as NMR becomes a more mature science, there’s always a risk that the rate of progress can slow down: “To keep the subject evergreen and continuing to offer benefits to society, we must continually strive to achieve more. In this regard, it’s very rewarding to work with Bruker – they have first-rate scientists, excellent instruments, and their work is of the highest standard. Together, we solve new problems, and keep each other at the forefront of science. They truly are ‘experts among experts’.”

Serendipity, curiosity, and shared goals
Another theme that Prof. Frydman is keen to emphasise is the value of collaboration in this field of science. “Covid-19 has provided a very strong reason to share data, results, and ideas, meaning that developments that would normally have taken years to come to fruition have happened much faster.” He adds that this co-operation comes quite naturally: “Compared to other areas of research, NMR is actually quite a collegiate field. I think this transparency has been enhanced further in this consortium, and I hope that some of the ways in which we’ve been working over the last year will remain with us even as Covid-19 fades into the background.”
Nevertheless, he is realistic about what lies ahead: “Making progress on Covid-19 research will require plenty of hard work – and for me at least, strong magnets and a lot of data-processing. But we must also acknowledge the role of serendipity and curiosity in this area of research – we must be willing to take on board surprising observations, or to think about problems in unconventional ways. One of the wonderful things about the Covid-19 consortium is that it makes this much easier, because we have a defined goal in mind, and we are ready to share our best ideas to make progress.”

This point Prof. Frydman makes about collaboration links through to a final thought on what the last year has taught him: “We have a lot in common as scientists of course, but this virus has shown us that we also have a lot in common as human beings. So the way we’ve been working together within this consortium to study SARS-CoV-2 using NMR should inspire us to cooperate in other areas, to solve problems that affect everyone on the planet.”

For more information on the Covid19-NMR consortium, please visit https://covid19-nmr.de/ or watch the interviews with the consortium members https://www.bruker.com/en/products-and-solutions/mr/make-mr-more-relevant/covid19-nmr-consortium.html

For more information about the Frydman Group, please visit https://www.weizmann.ac.il/chembiophys/Frydman_group/  

Professor Lucio Frydman is head of the Department of Chemical and Biological Physics at the Weizmann Institute of Science in Rehovot, Israel, where he has been since 2001. He is also Chief Scientist in Chemistry and Biology at the National High Magnetic Field Laboratory in Tallahassee, Florida, USA.

He has published over 250 peer-reviewed papers, and from 2011-2020 he acted as Editor-in-Chief of the Journal of Magnetic Resonance. He is currently the founding editor of JMR – Open, a companion to the Journal of Magnetic Resonance.

1. For more about the work of the Prof. Frydman group, visit https://www.weizmann.ac.il/chembiophys/Frydman_group/home
2. For more about the Covid-19 consortium, visit https://covid19-nmr.de/
3. A. Wacker et al., Secondary structure determination of conserved SARS-CoV-2 RNA elements by NMR spectroscopy, Nucleic Acids Research, 2020, 48: 12415–12435.
4. S. Marković, A. Fages, T. Roussel, R. Hadas, A. Brandis, M. Neeman and L. Frydman, Maternal-fetal exchanges and placenta metabolism followed in real-time by dynamic 13C hyperpolarized MRSI, Proc. Natl. Acad. Sci. USA, 2018; 115 (10) E2429-E2436. 
   
5. M. Novakovic, G. Olsen, G. Pinter, D. Hymon, B. Fürtig, H. Schwalbe and L. Frydman, >300-fold enhancement of imino nucleic acid resonances via hyperpolarized water: A new window for probing RNA refolding by 1D and 2D NMR; Proc. Natl. Acad. Sci. USA, 2020; 117, 5, 2449-2455.
6. J. Kim et al., 3D Heteronuclear magnetization transfers for the establishment of secondary structures in SARS-CoV-2-derived RNAs, Journal of the American Chemical Society, 2021, 143: 4942–4948.
   [7] M. Novakovic et al., Magnetization transfer to enhance NOE cross-peaks among labile protons: Applications to imino-imino sequential walks in COVID-derived RNAs, Angewandte Chemie International Edition, 2021 (accepted article).
7. M. Novakovic, E. Kupce, A. Oxenfarth, M.D. Battistel, D.I. Freedberg, H. Schwalbe. and L. Frydman, Hadamard magnetization transfers achieve dramatic sensitivity enhancements in homonuclear multidimensional NMR correlations of labile sites in proteins, polysaccharides and nucleic acids, Nat Comm, 2020; 11, 5317 (2020).  
   
   

The Weizmann Institute in Rehovot, Israel, has been at the forefront of NMR research since 1952, when a group led by Saul Meiboom constructed one of the world’s first NMR spectrometers. There are now 12 groups working in NMR at the institute, in fields spanning everything from detection of single molecules through to functional MRI imaging of the brain.

For more information please visit: https://www.weizmann.ac.il/chembiophys/Frydman_group

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