MOLECULAR INSIGHTS INTO NUCLEIC ACID-MEDIATED PHASE SEPARATION OF PRION PROTEIN

The prion protein (PrP) is a GPI-anchored membrane protein predominantly expressed in neuronal cells, whose misfolding into a β-sheet-rich conformation (PrPSc) is associated with fatal neurodegenerative disorders known as Transmissible Spongiform Encephalopathies (TSEs). Neurodegenerative disease–associated proteins can undergo phase separation 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 five 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. The identified RNAs were further synthesized and purified as single-stranded DNA sequences and their ability to induce PrP aggregation and/or phase separation was evaluated using turbidity and light scattering assays, as well as microscopy techniques, including differential interference contrast (DIC) and fluorescence microscopy. The PrP–NA complexes formed spherical, micrometer-sized condensates. These condensates were highly sensitive to NaCl and to the aliphatic alcohol 1,6 hexanediol, suggesting that both electrostatic and hydrophobic interactions play important roles in the formation of the heteritypic condensates. Structural changes in rPrP induced by nucleic acid binding were evaluated by circular dichroism (CD) and intrinsic fluorescence spectroscopy. Alterations in the CD spectra were observed following NA interaction, indicating changes in the protein's secondary structure and highlighting the structural impact of PrP–NA interactions. Ultimately, this study aims to identify and functionally validate nucleic acid sequences that promote PrP misfolding and aggregation, providing potential molecular targets for therapeutic strategies against prion-related diseases