Abstract Background: My laboratory works at the interface of cell biology and nucleic acid chemistry to modulate translation, splicing, and transcription. Ideal projects have three features: 1) Novel oligonucleotides that allow us to probe the strengths and weakness of the chemical modifications; 2) Challenging nucleic acid structures or mechanisms for nucleic acid-mediated regulation; and 3) The opportunity to gain insights into significant biological process or therapeutic development. For 2021- 2026 our central goal is to investigate mechanisms of RNA recognition to gain greater insights into cell physiology and therapeutic development. Since 2016, several nucleic acid drugs have been approved and oligonucleotide medicine is poised to have a significant impact on broad patient populations. One obstacle to realizing this potential is an incomplete understanding of RNA recognition in cells. For example, while over 1000 peer-reviewed publications appear every month that cite the term “miRNA”, we lack the detailed insights necessary to necessary to predict function or appreciate the roles of RNA in complex biological processes. Validation of proposed effects is often problematic and the predictive power for studying novel genes is limited. We will focus on two project areas involving the recognition of RNA within either the nuclei or cytoplasm of human cells. Cooperation of miRNAs and protein factors during RNAi. We will gain fundamental insights into the mechanism of RNA using knock out cell lines deficient for key RNAi factors including proteins of the argonaute (AGO) and trinucleotide repeat containing protein 6 (TNRC6) families. We will combine mass spectrometry, RNAseq, and cross linking immunoprecipitation (CLIP) to define where interactions occur how the number of protein or miRNAs affect the impact on gene regulation. We will first examine detailed mechanisms for regulation in cell cytoplasm. We will then apply these lessons to investigate the consequences of miRNA recognition in both cell cytoplasm and nuclei. Novel mechanisms and disease targets. We will gain a better understanding of two diseases caused by mutant RNAs, Fuch's Corneal Endothelial Dystrophy (FECD) and Friedreich's Ataxia (FA). One goal is to develop more potent compounds to facilitate drug development. A second goal is to better understand mechanisms of action. Both diseases involve nuclear noncoding RNAs, and we will use synthetic nucleic acids as tools to probe disease mechanism and better appreciate how a small number of mutant RNA molecules can cause late-onset degenerative diseases.