Periodic Reporting for period 4 - PARAMIR (Investigating micro-RNA Dynamics using Paramagnetic NMR Spectroscopy)
Reporting period: 2023-08-01 to 2025-01-31
Over the last decades, ribonucleic acids (RNAs) have revolutionized our vision of biology. From being a simple messenger allowing to translate the deoxyribonucleic acid (DNA) genetic information into proteins, RNA is now a central player in biology. Among its numerous functions, RNA can accelerate chemical reactions, regulate genetic information or help in information transfer. RNA has recently been unveiled to the general audience due to Covid vaccines, however, it has been seen as a promising way for designing novel therapies since many years and will likely be the basis of major therapeutic breakthroughs in the next decades.
Despite the major fundamental and practical interest for RNA, the understanding of this molecule at the atomic level remains limited. How does an RNA structure itself? How does it move? Are RNA motions important and in which way for its biological function or for designing biotherapies?
Those questions are of major interests for the society. Among the three large classes of biomolecules (DNA, RNA and proteins) RNA remain the least understood. Looking back at the study of proteins, the fundamental understanding of those systems has paved the way of structural biology, allowing for the development of innovative therapies or for various bioengineering advances. Similarly, understanding the fundamental properties of DNA was a major leap forward, providing the molecular rational for heredity, and leading to the development of modern genomic and its numerous applications. The fundamental understanding of RNA is expected to provide as important, yet unknown, discoveries and applications.
If the current progress of biology is sketching RNA importance and functional diversity, the understanding of this key molecule in terms of physical chemistry is still lagging. One of the fundamental reasons explaining this delay, reside in the intrinsic difficulties in working with RNA and observing them at atomic resolution. The objective of the project is to develop a novel strategy based on Nuclear Magnetic Resonance (NMR) to unravel at an unprecedented level of detail the molecular flexibility of RNA molecules. This methodology will be first used to better understand the molecular basis of gene regulation by RNA before being used on various essential RNA. This would thus help in the fundamental understanding of RNA physical chemistry and its connection with RNA function.
Despite the major fundamental and practical interest for RNA, the understanding of this molecule at the atomic level remains limited. How does an RNA structure itself? How does it move? Are RNA motions important and in which way for its biological function or for designing biotherapies?
Those questions are of major interests for the society. Among the three large classes of biomolecules (DNA, RNA and proteins) RNA remain the least understood. Looking back at the study of proteins, the fundamental understanding of those systems has paved the way of structural biology, allowing for the development of innovative therapies or for various bioengineering advances. Similarly, understanding the fundamental properties of DNA was a major leap forward, providing the molecular rational for heredity, and leading to the development of modern genomic and its numerous applications. The fundamental understanding of RNA is expected to provide as important, yet unknown, discoveries and applications.
If the current progress of biology is sketching RNA importance and functional diversity, the understanding of this key molecule in terms of physical chemistry is still lagging. One of the fundamental reasons explaining this delay, reside in the intrinsic difficulties in working with RNA and observing them at atomic resolution. The objective of the project is to develop a novel strategy based on Nuclear Magnetic Resonance (NMR) to unravel at an unprecedented level of detail the molecular flexibility of RNA molecules. This methodology will be first used to better understand the molecular basis of gene regulation by RNA before being used on various essential RNA. This would thus help in the fundamental understanding of RNA physical chemistry and its connection with RNA function.
The work performed focused on novel NMR strategies to study the conformational dynamics of flexible RNA and apply it to relevant RNA to understand to which extend their biology can be understood using physical chemistry.
First, the work focused on RNA synthesis needed to bypass current limitations of NMR, as RNA both isotopically labelled with NMR active nuclei and with site specific chemical modification to induce specific NMR effects are needed. This can be obtained combining strategies from both chemistry and molecular biology. We have developed those approaches in parallel and use biochemical tools to fuse them in a single molecule. We used this strategy to either introduce unique NMR active nuclei within the RNA molecule or to introduce a chemical group that will affect other NMR signals, and thereby generates new NMR datasets. We have worked in the perspective of targeting, complex, biologically relevant RNA, enforcing us to improve existing NMR methodologies to access such systems.
Second, important efforts were dedicated to decode the information obtained by NMR into realistic model of motions. For that, we extended existing methodologies to analyze various diamagnetic and paramagnetic NMR data. We rationalized known discrepancies between NMR and Small Angle X-ray Scattering (SAXS) and propose a framework to interpret them jointly. The study of RNA flexibility is often limited to model systems also by the difficulty to model large conformational changes in RNA. Starting with state-of-the-art modelling approaches to probe slow timescale motions, and refining it with our data, we could propose a description at high resolution of RNA motions significantly larger than usually studied. We also proposed an alternate strategy to decipher RNA conformational dynamics in absence of potential bias arising from classical numerical simulations, which is essential to deeply understand RNA intrinsic properties.
Finally, these methodologies were applied to a series of systems, allowing to shed light on the conformational dynamics of various non-coding RNA. We worked on the micro-RNA (miRNA) let-7 and demonstrate that we can probe its conformational properties in interaction with its biological targets using fluorine probes. We also worked on the preliminary miRNA let-7 and we could deconvolute its complex dynamics. This suggests a mechanism in which its intrinsic plasticity allows for a molecular switch that explains the competitive regulation by two biological partners. We could also shed light on the fuzzy interaction between an RNA and an intrinsically disordered protein and help in understanding the basis of liquid-liquid phase separation. We also demonstrated the interest of these methodologies to study other key RNA by investigating the multi-timescale dynamics of transfer RNA at atomic resolution.
While this project was focusing on basic science, the skills and knowledge accumulated during this work allowed us to propose a novel strategy to target emerging viruses, that we could demonstrate to be efficient in vitro.
First, the work focused on RNA synthesis needed to bypass current limitations of NMR, as RNA both isotopically labelled with NMR active nuclei and with site specific chemical modification to induce specific NMR effects are needed. This can be obtained combining strategies from both chemistry and molecular biology. We have developed those approaches in parallel and use biochemical tools to fuse them in a single molecule. We used this strategy to either introduce unique NMR active nuclei within the RNA molecule or to introduce a chemical group that will affect other NMR signals, and thereby generates new NMR datasets. We have worked in the perspective of targeting, complex, biologically relevant RNA, enforcing us to improve existing NMR methodologies to access such systems.
Second, important efforts were dedicated to decode the information obtained by NMR into realistic model of motions. For that, we extended existing methodologies to analyze various diamagnetic and paramagnetic NMR data. We rationalized known discrepancies between NMR and Small Angle X-ray Scattering (SAXS) and propose a framework to interpret them jointly. The study of RNA flexibility is often limited to model systems also by the difficulty to model large conformational changes in RNA. Starting with state-of-the-art modelling approaches to probe slow timescale motions, and refining it with our data, we could propose a description at high resolution of RNA motions significantly larger than usually studied. We also proposed an alternate strategy to decipher RNA conformational dynamics in absence of potential bias arising from classical numerical simulations, which is essential to deeply understand RNA intrinsic properties.
Finally, these methodologies were applied to a series of systems, allowing to shed light on the conformational dynamics of various non-coding RNA. We worked on the micro-RNA (miRNA) let-7 and demonstrate that we can probe its conformational properties in interaction with its biological targets using fluorine probes. We also worked on the preliminary miRNA let-7 and we could deconvolute its complex dynamics. This suggests a mechanism in which its intrinsic plasticity allows for a molecular switch that explains the competitive regulation by two biological partners. We could also shed light on the fuzzy interaction between an RNA and an intrinsically disordered protein and help in understanding the basis of liquid-liquid phase separation. We also demonstrated the interest of these methodologies to study other key RNA by investigating the multi-timescale dynamics of transfer RNA at atomic resolution.
While this project was focusing on basic science, the skills and knowledge accumulated during this work allowed us to propose a novel strategy to target emerging viruses, that we could demonstrate to be efficient in vitro.
First, we provided the first high-resolution description of a large RNA loop conformational dynamics. This was possible by combining state-of-the-art biochemical methodologies, ultra-high-field NMR spectroscopy and advanced modelling. This result is essential as it shed a new light on the biophysical mechanism underlying complex biological regulatory pathways. Additionally, by demonstrating the feasibility of such study, this will become a driving force to study other challenging but essential RNA.
Second, we proposed a novel methodology to fight emerging viruses using rationally designed NMR optimized small interfering RNA (siRNA). We could demonstrate in vitro their efficiency against the SARS-CoV-2 and influenza. This could potentially be the basis of future innovative therapies.
Third, we could propose the first detailed description of an interaction between an RNA and an unfolded protein, something usually not studied due to the complexity of accumulating detailed data on these systems. Our results help in understanding liquid-liquid phase separation process, a biophysical mechanism allowing to regulate biomolecular activity.
Fourth, the methodology based on fluorine offers a novel avenue to study conformational dynamics of miRNA or siRNA in interaction with their biological targets, that should become particularly attractive to study this process in close to physiological conditions or even in the cell.
Finally, we have proposed a set of new methodologies that taken together open new opportunities to study conformational dynamics of complex RNA. These methodologies are at the crossing of NMR, biochemistry, molecular biology, synthetic and theoretical chemistry and biophysics. They will allow to measure data on challenging systems, access new NMR probes and interpret simultaneously large experimental datasets in light of advanced modelling. We expect them to help in the future to study challenging RNA molecules, alone or in interaction.
Second, we proposed a novel methodology to fight emerging viruses using rationally designed NMR optimized small interfering RNA (siRNA). We could demonstrate in vitro their efficiency against the SARS-CoV-2 and influenza. This could potentially be the basis of future innovative therapies.
Third, we could propose the first detailed description of an interaction between an RNA and an unfolded protein, something usually not studied due to the complexity of accumulating detailed data on these systems. Our results help in understanding liquid-liquid phase separation process, a biophysical mechanism allowing to regulate biomolecular activity.
Fourth, the methodology based on fluorine offers a novel avenue to study conformational dynamics of miRNA or siRNA in interaction with their biological targets, that should become particularly attractive to study this process in close to physiological conditions or even in the cell.
Finally, we have proposed a set of new methodologies that taken together open new opportunities to study conformational dynamics of complex RNA. These methodologies are at the crossing of NMR, biochemistry, molecular biology, synthetic and theoretical chemistry and biophysics. They will allow to measure data on challenging systems, access new NMR probes and interpret simultaneously large experimental datasets in light of advanced modelling. We expect them to help in the future to study challenging RNA molecules, alone or in interaction.