Improvement of the NMR structural quality for RNA and DNA

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Nuclear Magnetic Resonance spectroscopy (NMR) is very important for the characterization of DNA and RNA, encompassing a variety of methods that offer essential insight on the structure, function, interactions and conformational dynamics of these biomolecules. Despite the eminent contribution of NMR in this field, some essential tools and approaches (which are routinely available for proteins) are still missing for nucleic acids and require further development or fine-tuning. Notably, the parametrization of forcefields used for the structure determinations of nucleic acids still lacks behind. Advancing the development in this area is therefore indispensable to elucidate more accurate and high-quality well resolved NMR structures. Such approach requires verification with well characterized RNA and DNA reference systems. Our objective is to improve the currently established methods and protocols and provide high quality data to optimize the forcefield parametrization. In this respect, we already elucidated the structure of two RNA hairpin model systems (the tetraloops UUCG and CUUG) using a wealth of structural data and incorporated optimized potentials for refinement in water. The UUCG structure is currently considered to be the gold standard for forcefield optimizations and assessment by cross-validation with other methods and empirical data. We currently investigate the structure and dynamics of additional tetraloops (GCAA, GAAG). Moreover, we provide additional suitable model targets from our current extensive investigations of elements from the genome of RNA viruses (Covid-19 and West Nile Virus) and other small DNA and RNA motifs and repeats. In our endeavor to describe the ensemble characteristic of RNA structures realistically, we studied the CUUG tetraloop system, which turned out to feature substantial dynamics despite its high thermal stability. Thus, we have recently combined NMR with molecular dynamic simulations to probe the underlying conformational landscape and provide suitable structural ensembles. In this application, we will include larger RNA and DNA molecules, G-Quadruplexes, and RNA/DNA triplexes. For their structure determination, we will gather additional global, long-range structural, orientational and dynamic restraint data and utilize 3D structure prediction and homology modeling for evaluation of the nucleic acid forcefield and improvement of tools and protocols. Moreover, we aim to include the most important and occurring RNA and DNA modifications as well as several ions and plan to employ and develop approaches for the structure calculation of conformational ensembles. These new and improved parameters and methods will be made available for the structural NMR community in established software and through implementation in our existing web-portal service for NMR structure determination. In addition, we will publish the results in suitable open source databases, journals and platforms.