Protein-RNA interactions (PRIs) control pivotal steps in RNA biogenesis, regulate multiple physiological and pathological cellular networks, and are emerging as important drug targets. However, targeting of specific protein-RNA interactions for therapeutic developments is still poorly advanced. Studies and manipulation of these interactions are technically challenging and in vitro drug screening assays are often hampered due to the complexity of RNA structures. The binding of nucleolin (NCL) to a G-quadruplex (G4) structure in the messenger RNA (mRNA) of the Epstein-Barr virus (EBV)-encoded EBNA1 has emerged as an interesting therapeutic target to interfere with immune evasion of EBV-associated cancers. Using the NCL-EBNA1 mRNA interaction as a model, we describe a quantitative proximity ligation assay (PLA)-based in cellulo approach to determine the structure activity relationship of small chemical G4 ligands. Our results show how different G4 ligands have different effects on NCL binding to G4 of the EBNA1 mRNA and highlight the importance of in-cellulo screening assays for targeting RNA structure-dependent interactions.

Chemistry Modelling and Imaging for Biology CNRS UMR9187 Inserm U1196 Institut Curie Université Paris Sud F 91405 Orsay France

GGB Université de Brest Inserm CHRU Brest EFS UMR 1078 F 29200 Brest France

Université Paris 7 Inserm UMR 1162 75013 Paris France

Université Paris 7 Inserm UMR 1162 75013 Paris France ICCVS University of Gdańsk Science ul Wita Stwosza 63 80 308 Gdańsk Poland

Université Paris 7 Inserm UMR 1162 75013 Paris France ICCVS University of Gdańsk Science ul Wita Stwosza 63 80 308 Gdańsk Poland Department of Medical Biosciences Umeå University 90187 Umeå Sweden RECAMO Masaryk Memorial Cancer Institute Zluty kopec 7 65653 Brno Czech Republic

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