MOLECULAR INSIGHTS INTO NUCLEIC ACID-MEDIATED PHASE SEPARATION OF PRION PROTEIN AND ITS CONTRIBUTION TO PRION DISEASE

The prion protein (PrP) is a GPI-anchored membrane protein predominantly expressed in neuronal cells, whose misfolding into a β-sheet-rich conformation (PrP^Sc) is associated with fatal neurodegenerative disorders known as Transmissible Spongiform Encephalopathies (TSEs). Neurodegenerative disease–associated proteins can undergo liquid–liquid phase separation (LLPS) to form biomolecular condensates, which are thought to represent precursors of neurotoxic protein aggregates through a liquid-to-solid transition. For the prion protein (PrP), both homotypic LLPS (involving only PrP) and heterotypic LLPS (involving PrP and a ligand, such as nucleic acids) have been reported. This project aims to characterize RNA sequences from murine neuroblastoma (N2a) cells that were previously identified, through next-generation sequencing, as binding partners of recombinant murine PrP (rPrP) capable of promoting its aggregation in vitro. A total of 64 RNA sequences derived from RNA-seq and ChIP-seq experiments were analyzed, and their secondary structures were predicted using bioinformatics algorithms. Total RNA extracts from N2a cells will be analyzed by circular dichroism (CD) and isothermal titration calorimetry (ITC), both untreated and treated with RNase A, to evaluate the contribution of RNA integrity to PrP binding and structural changes. The ability of these RNAs to induce PrP aggregation and/or liquid–liquid phase separation (LLPS) will be assessed by turbidity and light scattering assays, as well as microscopy techniques including transmission electron microscopy (TEM), differential interference contrast (DIC), and fluorescence microscopy. Structural changes in rPrP upon nucleic acid binding will also be monitored by CD and intrinsic fluorescence spectroscopy. In addition, specific RNA sequences enriched in the total RNA extract of N2a cells are being synthesized. These molecules will be investigated for their interaction with rPrP using ITC, fluorescence anisotropy, and fluorescence microscopy, and will also be subjected to CD to determine their binding parameters and structural effects. Ultimately, this study aims to identify and functionally validate nucleic acid sequences that facilitate PrP misfolding and aggregation, providing potential molecular targets for therapeutic strategies against prion-related diseases.