Periodic Reporting for period 1 - HRRinDNAwithSSB (Unraveling nanosecond motions in nucleic acids with high-resolution relaxometry: how dynamic is nicked DNA?)
Okres sprawozdawczy: 2021-09-01 do 2023-08-31
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.
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.