Pseudocontact chemical shifts in RNA via lanthanide chelate tagging

1.Summary of the research plan1.1.Background and rationaleRibonucleic acids (RNA) play crucial roles in many cellular processes. A mandatory pre-requisite to understand the biological function of RNAs is the availability of high-resolution structures together with the knowledge of the functional dynamics. Pseudo-contact shifts (PCSs) are an important source of information to address functional features of a biomolecule by attaching lanthanide chelating tags (LCTs). There are numerous lanthanide-based tags for measuring PCSs in proteins, for RNA and DNA such modifiers are still not widely disseminated. Thus, an approach that allows direct RNA lanthanide chelate tagging would be highly desirable.1.2.Overall objectivesThe objective of the research is the expansion of the NMR toolbox for nucleic acids by the introduction of LCTs to modify pre-functionalized RNAs carrying 13C/15N-stable isotope labels.1.3.Specific aims- A synthetic access to a position 5-modified pyrimidine RNA building blocks for post-synthetic RNA modification (Kreutz Group).- Incorporation of the 5-modified pyrimidine building blocks together with stable isotope labelled residues into RNAs up to 64 nucleotides (nts) (Kreutz Group).- A synthetic access to isothiocyanate and azide DOTA-M7-tag (Häussinger Group).- Establishing a direct and reliable lanthanide chelate tagging protocol for RNA comprising up to 64 nts (Häussinger and Kreutz Group).- Combining the direct RNA LCT protocol with 13C/15N stable isotope labelling by solid phase RNA synthesis to extract PCSs and RDCs in RNA (Häussinger and Kreutz Group).1.4.MethodsOrganic synthetic chemistry and biomolecular solution NMR spectroscopy will be used as the main methods.1.5.Expected resultsThe research will close the currently existing gap between protein and RNA/DNA paramagnetic nuclear magnetic resonance (NMR) spectroscopy. The anticipated protocols from this research will result in a user-friendly protocol for direct lanthanide tagging of RNAs up to approximately 70 nts. This is especially interesting for the class of non-coding RNAs with this size, such as riboswitch aptamers or pre-micro RNA, and to answer biologically relevant open questions of the function of these RNAs. The protocols will allow to extract accurate distance information in RNA, and in RNA-RNA, RNA-small molecule and RNA-protein complexes. The RNA DOTA tagging will further open new possibilities in RNA drug targeting and to study functional RNA dynamics.1.6.Impact for the scientific field and beyondThe proposed research project will further reduce the methodological gap between protein and nucleic acid NMR spectroscopy and will allow to gain insights into the structure and function of larger RNAs and large RNA protein complexes. This is of special importance as non-coding RNAs and RNA protein complexes get more and more recognized as a drug target and sophisticated NMR methods will be a key technology in drug screening processes.