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“We are often charged with not being sensitive enough,” says Prof. Lucio Frydman of the Weizmann Institute of Science’s Chemical and Biological Physics Department. Frydman is not referring to emotional detachment but rather to nuclear magnetic resonance (NMR), a tool used to elucidate, with atomic-level resolution, the structure and dynamics of molecules. Although used around the world in academic biological and chemical research and in the pharmaceutical industry, NMR is notorious for the weakness of its signals. Working with researchers in Frankfurt, Germany, Frydman and his group developed an NMR method for studying biomolecules at an atomic level under physiological conditions; this method is hundreds of times more sensitive than the usual ones – enough so to catch dynamic biomolecular rearrangements at concentrations that are significantly lower than those normally assayed. The researchers hope that their development will advance the study of biomolecules, particularly RNA and proteins.
Each time a biomolecule gets a polarized nucleus from the water, it can emit a “super-signal” that is hundreds of times more intense than a regular NMR response
Like the more familiar MRI, which is a particular kind of NMR scan, NMR utilizes magnetic fields to align and then track the behavior of atomic nuclei. Magnetic interactions, however, are conspicuously weak. While this is a great feature that makes MRI remarkably noninvasive, it robs NMR of some much-needed sensitivity. One of the avenues researchers have been exploring for increasing sensitivity, explains Frydman, is hyperpolarization – a means of greatly increasing the number of spins that become aligned in the sample. But nuclear hyperpolarization usually requires cryogenic operations that do not work well for molecules such as RNA, DNA or proteins – particularly if one’s aim is to study these under their natural, physiological conditions. Over the past few years, Frydman and his group have tried to approach the problem from a new angle: to hyperpolarize the hydrogen nuclei of water molecules. This can be done at a temperature close to absolute zero – at which, even under weak fields, the nuclei can align very well. The trick is to melt this water all at once and “shower” it onto a tube where the biomolecules to be studied are waiting within a conventional NMR setup. Under suitable conditions, the water’s hydrogen atoms can then spontaneously exchange with their counterparts in the biomolecules – quickly bouncing back and forth between the water’s oxygen and nitrogen atoms in the RNA bases or amide bonds in proteins. This shuffling does not affect the hydrogen atoms’ nuclear alignment: Each time a biomolecule gets a polarized nucleus from the water, it can emit a “super-signal” that is hundreds of times more intense than a regular NMR response. Frydman, together with graduate student Mihajlo Novakovic and senior intern Dr. Gregory Olsen, exploited this, “filming” the dynamics of molecules known as riboswitches – bits of messenger RNA that rapidly flip from one state to another when activated by the binding of a small molecule – at a rate of around three frames per second.
“The important thing we learned is: ‘don’t touch the water’ once it has been injected into the sample,” says Frydman. “The water acts as a sort of bank of hyperpolarized nuclei for the biomolecules, which can thus be investigated over the course of a minute or two with various NMR approaches.”
“Other techniques get information about structures or about dynamic intermediates by cooling them, and consequently slowing down their movements so they can be observed; by contrast our experiments can be conducted at physiological temperatures and in their natural milieu,” says Novakovic. “We were even able to note which parts of the RNA were more active and which remained sheltered from interactions, by observing how facile their exchanges were with the hyperpolarized water,” he adds.
To conduct these experiments, Novakovic, Olsen and Frydman used two NMR machines at the Weizmann Institute of Science. One, working at ultracold temperatures, was used to hyperpolarize the water, which was then quickly warmed and injected by a computer-controlled device built for the purpose – together with the molecule that induced the riboswitch’s conformational change – into a second NMR system that held the RNA molecule at physiological conditions. The design of this RNA experiment, together with the preparation of the samples, was conducted in partnership with Frydman’s German-Israeli Foundation collaborators at the Johann Wolfgang Goethe University in Frankfurt, led by Prof. Harald Schwalbe.
Now that they have shown the benefits of using hyperpolarized water for unraveling RNA dynamics, the group intends to keep developing the method. In particular these researchers are extending these NMR experiments, which were recently described in the Proceedings of the National Academy of Sciences (PNAS), USA, to more complex biomolecules, including other RNAs and proteins, as well as to experiments achieving higher levels of hyperpolarization.
Prof. Lucio Frydman is Head of the Helen and Martin Kimmel Institute for Magnetic Resonance Research; the Clore Institute for High-Field Magnetic Resonance Imaging and Spectroscopy; and the Fritz Haber Center for Physical Chemistry. His research is supported by the Leona M. and Harry B. Helmsley Charitable Trust; the Dr. Dvora and Haim Teitelbaum Endowment Fund; and the Harold Perlman Family. Prof. Frydman is the incumbent of the Bertha and Isadore Gudelsky Professorial Chair.