Probing the functional dynamics of protein(/metalloprotein)-nucleic acid interactions for potential applications in chemical biology Dr Somnath Mondal, Department of Chemistry, Pennsylvania State University, USA The picornaviral 3C(D) [PV-3C(D)] protein is a crucial protease that cleaves the viral polyprotein and host cell defense proteins1 . This protein also interacts with cis-acting replication elements (CREs) to control viral replication and translation, especially essential in the timing of the viral life cycle2-3. However, the molecular details of the 3C(D) and RNA interaction are poorly understood. As such, we have studied the sequence and structure determinants of RNA binding to PV-3C(D) using a range of biophysical techniques including NMR, AUC, SAXS, and DIC microscopy. These studies have indicated that PV-3C(D) binds a wide range of RNA with little to no sequence and structure dependence. Surprisingly, these studies have also indicated that PV3C(D)-RNA interactions lead to liquid-liquid phase separation (LLPS) [Figure 1]. LLPS has been previously observed in other viral infections, which may play a role in virally induced apoptosis and virus spread. A similar finding with 3C(D) opens yet another function for this protein central to the viral life cycle. Interactions between metalloproteins and nucleic acids also play important roles in DNA repair and mitochondrial dysfunction. Mitochondrial dysfunction, a hallmark of aging in long-lived animals, is caused by accumulated mitochondrial DNA damage. Octocorals, a mitochondrially encoded MutS protein, have the required endonucleolytic activity to bind and cleave DNA for DNA damage repair4 . Endonucleases cleave DNA internally, and do not require free DNA ends for activity. We expressed the isolated domains from D. gigantea and consensus sequence from available mt-MutS proteins showing endonuclease activity towards plasmid and linear DNA. Our further studies are being conducted to characterize and study DNA binding on various mutants of the C-terminal endonuclease domain utilising advanced biophysical techniques, bioinformatics, and AI. This shows the C-terminal domain is Zn+2 bound and has an effect on endonucleolytic activities upon the addition of various metal-ions. The study provides a detailed insight into DNA mismatch repair by the C-terminal domain of mt-MutS metalloprotein to understand the aging process in long-lived animals.