The Mesoscale Biophysics of RGG/RNA Interactions

This project addresses fundamental questions about how proteins interact with ribonucleic acid (RNA) molecules to regulate critical cellular processes. The second most common RNA-binding domain in the human proteome is the arginine-glycine-glycine (RGG) motif, which is present in more than 1,000 human proteins involved in all levels of RNA metabolism. Despite its prevalence, how the RGG motif functions remains poorly understood because of its highly disordered and dynamic nature, which prevents study by traditional structural biology techniques. Consequently, a clear understanding of how these motifs recognize RNA, or contribute to the formation of liquid droplets inside cells, is currently lacking. By resolving these fundamental mechanisms, this project advances the national interest, health, and welfare, as the dysfunction of RGG-containing proteins is implicated in human disease. Furthermore, the conceptual advances from this research will benefit society and biotechnology by informing the design of responsive biomaterials with programmable properties and synthetic protocells. The project also directly contributes to STEM education and workforce development by providing graduate and undergraduate students with hands-on training in single-molecule instrumentation and biochemistry. The technical objective of this project is to establish the quantitative physical principles governing mesoscale (10–25 residues) RGG/RNA interactions using a closely integrated approach that combines single-molecule mechanical manipulation, bulk phase behavior, and computational modeling. Specifically, the project investigates how sequence features, including RGG valence and aromatic residue content, along with ionic conditions dictate binding affinities and structural outcomes. Using a custom-built magnetic tweezers platform, the researchers will apply stable forces to single RNA molecules to track, in real time, how RGG peptides modulate local RNA flexibility, base-stacking, and force-dependent structural transitions. The research encompasses both structured and unstructured RNAs, thus directly testing the relative importance of non-specific electrostatic screening and structure-specific interactions. Single-molecule experiments will be paired with bulk turbidity measurements to correlate nanoscale interaction strengths with macroscopic liquid-liquid phase separation. Concurrently, atomistic and coarse-grained molecular simulations will provide mechanistic insight into the electrostatic and non-electrostatic components of the interaction. Ultimately, this work will generate a predictive framework for disordered protein-nucleic acid interactions, resolve paradoxes regarding whether RGG motifs stabilize or destabilize RNA structures, and supply high-quality benchmark datasets to refine multi-scale computational biophysics models. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria. NSF Award ID: 2610165 | Program: 01002627DB NSF RESEARCH & RELATED ACTIVIT | Principal Investigator: Omar Saleh | Institution: University of California-Santa Barbara, SANTA BARBARA, CA | Award Amount: $967,237 View on NSF Award Search: https://www.nsf.gov/awardsearch/show-award/?AWD_ID=2610165 View on Research.gov: https://www.research.gov/awardapi-service/v1/awards/2610165.html