The increasing awareness of the essential role of RNA in biological processes and progression of diseases, including its involvement in translation, gene regulation, and viral infections, make RNA an interesting target for therapeutic intervention. Riboswitches or the tmRNA-mediated rescue mechanism represent very attractive novel therapeutic approaches against pathogenic bacteria, especially in light of the growing threat of emerging antibiotic resistance. Genomic RNA of pathogenic viruses such as HIV or SARS provides many opportunities for drug discovery. However, the discovery of new RNA-binding drugs has significantly lagged behind their protein targeting counterparts. This can be at least partially attributed to a paucity of state-of-the-art NMR methods to detect such interactions. RNA-ligand recognition typically occurs by conformationally flexible """"""""induced-fit"""""""" rather than by rigid """"""""lock-and-key"""""""" docking. The resulting greater disorder and the limited chemical diversity in comparison to proteins complicate NMR (and other) studies of RNA and its complexes. It is the goal of our application to establish a combination of NMR methods and RNA labeling techniques to facilitate the identification of specific, high affinity RNA-ligand interactions. A wellstudied example, HIV TAR, will serve as our model system. The Tat-TAR complex formation provides an essential gene regulatory function for HIV.
Our specific aims are: 1. To develop sensitive ligand-based NMR screening approaches utilizing 19F-labeled RNA We aim to explore heteronuclear 19F,1H-NOE interactions in NMR-based RNA-ligand binding studies. We propose to utilize chemically induced dynamic nuclear polarization (CIDNP) enhancements to boost sensitivity in this ligand-detected screening method. We will pursue CIDNP to facilitate the NMR analysis of 19F-labeled RNA in general. 2. To optimize and apply SAR by NMR follow-up validation assays using 19F-labeled RNA We will use 5-Fluoropyrimidine (major groove reporter) or 2-Fluoroadenine (minor groove reporter) substituted RNA in follow-up screening assays utilizing extraordinarily sensitive 19F-NMR chemical shift perturbations.
We aim to develop and apply segmental isotopic labeling approaches using T4 RNA ligase to overcome potential limitations associated with uniform 19F-labeling schemes. Public Health Relevance Statement: Many RNA molecules are drug targets with great potential for therapeutic treatment of bacterial or viral infections. However, the discovery of new RNA-binding drugs has been lagging because of the chemical properties of RNA and a lack of state-of-the-art technology to identify RNA-drug interactions. If the premises of our proof-of-concept studies are realized, then our approach will greatly facilitate the discovery of RNA-binding therapeutic candidates.
|Scott, Lincoln G; Hennig, Mirko (2016) ¹?F-Site-Specific-Labeled Nucleotides for Nucleic Acid Structural Analysis by NMR. Methods Enzymol 566:59-87|