C1 - Trityl Spin Labels: Developing Labeling Strategies for in cellulo and Temperature Dependent Pulsed Dipolar EPR Measurements on Nucleic Acid Complexes

Structural biology is engaging ever larger complexes and realized that the determined in vitro structures of such complexes may not be the functionally active ones within cells. Pulsed Dipolar EPR spectroscopy (PDS) in combination with site-directed spin labelling is a powerful method for providing access to biomacromolecular structures in vitro and within cells. However, for in-cell studies, the commonly used nitroxide labels for nucleic acids degrade very quickly. More stable labels are used either in combination with long, flexible linker groups and/or labelling strategies that only label the ends of nucleic acids. Here, we propose to develop strategies for site-specific internal labelling of nucleic acids with short-linked trityl radicals. We chose trityl radicals because they provide high sensitivity in PDS measurements and display high stability within cells. In addition, their long T_m relaxation times open a way to follow, e.g., ligand-binding induced structural changes in nucleic acid complexes from cryogenic temperatures up to room temperature. To translate the experimental data into structural models, extract thermodynamic and kinetic parameters, and gain mechanistic insights into the dynamics of nucleic acid complexes, we will complement our approach with all-atom molecular dynamics simulations (MD).