EFFECTS OF ANIONIC COMPOUNDS ON THE FORMATION AND STABILITY OF LIQUID CONDENSATES FORMED BY LIQUID–LIQUID PHASE SEPARATION MEDIATED BY THE N-NTD/SR DOMAIN OF THE SARS-COV-2 NUCLEOPROTEIN

SARS-CoV-2, the etiological agent responsible for COVID-19, remains a major public health challenge, being one of the main causes of acute respiratory infections and exhibiting a high capacity for adaptation and continuous emergence of viral variants. Among the structural proteins of the virus, the nucleoprotein (N) plays a central role in the replicative cycle, organizing the viral genomic RNA and ensuring the efficiency of replication and transcription. Recent evidence has shown that the N protein, particularly its N-terminal domain associated with an intrinsically disordered region rich in serine and arginine residues (N-NTD/SR), can form liquid condensates via liquid–liquid phase separation (LLPS) upon interacting with RNA. This mechanism promotes the compartmentalization and concentration of essential components for viral replication. Studies in the literature have demonstrated that the LLPS process of N-NTD/SR is governed by general physicochemical principles of electrostatic interactions. In this context, the present work aims to investigate the effect of different anionic compounds, including heparin, enoxaparin, suramin, chondroitin A, and chondroitin C, on the formation and stability of liquid condensates mediated by the N-NTD/SR of SARS-CoV-2. LLPS was assessed using UV–Visible spectroscopic turbidimetry and will be further investigated by bright-field microscopy, allowing the visualization and quantitative analysis of liquid condensates under different experimental conditions. Preliminary turbidimetry results show the formation of liquid condensates in the presence of 10% PEG (w/v) and at an RNA-to-protein molar ratio of approximately 7:1, with diminished condensate formation in the presence of heparin, indicating that N-NTD/SR condensate formation can be modulated by anionic compounds. These findings provide preliminary evidence for the proposed experimental approach and support further investigation of the effects of heparin and other anionic compounds on N-NTD/SR-mediated LLPS.