Periodic Reporting for period 1 - HRRinDNAwithSSB (Unraveling nanosecond motions in nucleic acids with high-resolution relaxometry: how dynamic is nicked DNA?)
Période du rapport: 2021-09-01 au 2023-08-31
Nucleic acids are vital for cellular life and have been shown to have a variety of functions ranging from information storage and transmission to catalysis. Central to their activity are the structures and dynamics they undergo. Understanding these processes is vital for characterising diseased states and the development of new therapeutics for a wide range of conditions from cancer to viral infections.
Typically, nucleic acids are structurally characterized using X-ray crystallography, Cryo-Electron Microscopy (Cryo-EM), and high-field Nuclear Magnetic Resonance (NMR). However, none of these approaches are suitable for studying nanosecond time-scale motions.
To address this issue, we will introduce a new method called High Resolution Relaxometry (HRR) to characterise dynamics in nucleic acids and apply it to a DNA helix. We expect that this will be the first general approach to obtain both time-scales and amplitudes for motions in both RNAs and DNAs. In conjunction to this we will carry our long molecular dynamics simulations to interpret our low field relaxation rates and provide an atomistic picture.
Typically, nucleic acids are structurally characterized using X-ray crystallography, Cryo-Electron Microscopy (Cryo-EM), and high-field Nuclear Magnetic Resonance (NMR). However, none of these approaches are suitable for studying nanosecond time-scale motions.
To address this issue, we will introduce a new method called High Resolution Relaxometry (HRR) to characterise dynamics in nucleic acids and apply it to a DNA helix. We expect that this will be the first general approach to obtain both time-scales and amplitudes for motions in both RNAs and DNAs. In conjunction to this we will carry our long molecular dynamics simulations to interpret our low field relaxation rates and provide an atomistic picture.
This project has been divided into three phases. In the first phase, we explored biochemical strategies for nucleic acid synthesis and prepared unlabeled samples for our relaxometry experiments.
In the second phase we developed experiments to measure longitudinal carbon relaxation rates in nucleic acids by shuttling the sample to different locations in the stray field. Here we showed that we could measure longitudinal relaxation rates for fields as low as 2 T in a site specific manner in a natural abundance sample. These measurements complimented by traditional R1, R2 and heteronuclear NOE experiments carried out at 500 MHz, 600 MHz, 800 MHz and 1GHz. In addition to this we developed a theoretical framework to interpret the entire experimental dataset using a model free formalism.
In the final phase these relaxation rates were interpreted using molecular dynamics simulations totalling over 68 microseconds. In this phase we were able to assess how well each other 3 force fields and 3 water models were able to reproduce the relaxation rates we recorded and allowed to evaluate how well each forcefield reproduced the kinetics and thermodynamic properties of the DNA helix. By doing so we gained insight into which motions give rise to relaxation processes.
In the second phase we developed experiments to measure longitudinal carbon relaxation rates in nucleic acids by shuttling the sample to different locations in the stray field. Here we showed that we could measure longitudinal relaxation rates for fields as low as 2 T in a site specific manner in a natural abundance sample. These measurements complimented by traditional R1, R2 and heteronuclear NOE experiments carried out at 500 MHz, 600 MHz, 800 MHz and 1GHz. In addition to this we developed a theoretical framework to interpret the entire experimental dataset using a model free formalism.
In the final phase these relaxation rates were interpreted using molecular dynamics simulations totalling over 68 microseconds. In this phase we were able to assess how well each other 3 force fields and 3 water models were able to reproduce the relaxation rates we recorded and allowed to evaluate how well each forcefield reproduced the kinetics and thermodynamic properties of the DNA helix. By doing so we gained insight into which motions give rise to relaxation processes.
This project goes beyond the state of the art by developing new approaches to characterise fast motions in nucleic acids and covering a range of timescales that are mostly missed by common techniques in structural biology. By developing these new methods for characterising motions in nucleic acids we hope to provide a deeper understanding of fundamental biological processes and open new routes targeting these systems with future therapeutics.