PROJECT SUMMARY / ABSTRACT If a facile method to site-selectively install prosthetic groups at internal sites in genetically-encoded RNA were available, then it would be possible to modify RNAs and ribonucleoprotein complexes (RNPs) in native structural and intracellular context, thus elevating studies on RNA folding, trafficking, lifetime, interactomes and regulatory pathways. The objective of this application is to test the extent to which compact U-rich internal loop (URIL) sites can be used as a general targeting motif in structured RNAs. If it were possible to selectively target the URIL motif with chemical probes, then juxtaposition of the URIL site with protein binding RNA motifs would enable tracking and chemical modification of ribonucleoprotein complexes (RNPs). This would enable elucidation of motif-specific RNA location and interactome by fluorogenic and proximity (biotin) labeling of URIL RNPs; such unbiased motif-centered interactome readout is not possible with existing methods. We hypothesize that appropriately modified, URIL-targeting bifacial peptide nucleic acids (bPNAs) could enable intracellular fluorogenic URIL (FLURIL) RNA tracking and proximity labeling of URIL (PLURIL) RNPs, respectively. Our proposed plan begins with the synthesis of bPNA probes, followed by rigorous in vitro and intracellular evaluation, optimization and validation with existing tools and known interactome partners. FLURIL RNP tagging will be benchmarked against MS2-labeling, the gold standard in RNA tracking. PLURIL tagging will be tested by its efficacy in identification of known RNPs. Further, we will test the extent to which URIL tags can be used to probe disease-relevant RNP biology in the intracellular context of amyotrophic lateral sclerosis (ALS), using patient-derived cells. Investigation of ALS pathology is a highly active area, with attention focused on two major forms: C9-ALS and Fus-linked ALS. While C9-ALS represents a majority of ALS cases, Fus-linked ALS is most commonly found in juvenile, aggressive early-onset cases; notably, the pathological mechanisms of these two forms appear to be distinct. Dysregulated RNP biology centered on C9orf72 RNA (C9-ALS) and U1snRNA (Fus-linked ALS) identifies these transcripts as prime substrates for URIL tag probes. The rigor in the prior research lies in the substantive preliminary and published data supporting intracellular fluorogenic and proximity labeling of URIL-RNPs. These data form a strong scientific premise for the impactful and unique application of motif-specific URIL-tagging as a broadly enabling discovery tool in ALS pathology and other RNP-centered diseases.