Pre-miRNA-149 G-quadruplex as a molecular agent to capture nucleolin

Autor: Tiago Santos, André Miranda, Lionel Imbert, Andreia Jardim, Catarina R.F. Caneira, Virgínia Chu, João P. Conde, Maria Paula Cabral Campello, António Paulo, Gilmar Salgado, Eurico J. Cabrita, Carla Cruz
Přispěvatelé: University of Beira Interior [Portugal] (UBI), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Instituto de Engenharia de Sistemas e Computadores Investigação e Desenvolvimento em Lisboa (INESC-ID), Instituto Superior Técnico, Universidade Técnica de Lisboa (IST)-Instituto de Engenharia de Sistemas e Computadores (INESC), Centro de Ciencias e Tecnologias Nucleares, Instituto Superior Tecnico, Universidade de Lisboa, Bobadela LRS, Portugal, Acides Nucléiques : Régulations Naturelle et Artificielle (ARNA), Université de Bordeaux (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), UCIBIO-REQUIMTE, Centro de Investigaçao Em Ciencias da Saúde - Health Sciences Research Center - University of Beira Interior (CICS-UBI), Centro de Investigação em Ciências da Saúde (CICS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Université de Bordeaux (UB), Thomas, Frank, UCIBIO - Applied Molecular Biosciences Unit, DQ - Departamento de Química
Jazyk: angličtina
Rok vydání: 2021
Předmět:
Zdroj: European Journal of Pharmaceutical Sciences
European Journal of Pharmaceutical Sciences, 2021, 169, pp.106093. ⟨10.1016/j.ejps.2021.106093⟩
European Journal of Pharmaceutical Sciences, Elsevier, 2021, 169, pp.106093. ⟨10.1016/j.ejps.2021.106093⟩
ISSN: 0928-0987
1879-0720
DOI: 10.1016/j.ejps.2021.106093⟩
Popis: PD/BD/142851/2018 PD/00065/2013 MIT-EXPL/BIO/0008/2017 IF/00959/2015 One of the most significant challenges in capturing and detecting biomarkers is the choice of an appropriate biomolecular receptor. Recently, RNA G-quadruplexes emerged as plausible receptors due to their ability to recognize with high-affinity proteins. Herein, we have unveiled and characterized the capability of the precursor microRNA 149 to form a G-quadruplex structure and determined the role that some ligands may have in its folding and binding capacity to nucleolin. The G-quadruplex formation was induced by K+ ions and stabilized by ligands, as demonstrated by nuclear magnetic resonance and circular dichroism experiments. Surface plasmon resonance measurements showed a binding affinity of precursor microRNA 149 towards ligands in the micromolar range (10−5–10−6 M) and a strong binding affinity to nucleolin RNA-binding domains 1 and 2 (8.38 × 10−10 M). Even in the presence of the ligand PhenDC3, the binding remains almost identical and in the same order of magnitude (4.46 × 10−10 M). The molecular interactions of the RNA G-quadruplex motif found in precursor miRNA 149 (5′-GGGAGGGAGGGACGGG- 3′) and nucleolin RNA-binding domains 1 and 2 were explored by means of molecular docking and molecular dynamics studies. The results showed that RNA G-quadruplex binds to a cavity between domains 1 and 2 of the protein. Then, complex formation was also evaluated through polyacrylamide gel electrophoresis. The results suggest that precursor microRNA 149/ligands and precursor microRNA 149/nucleolin RNA-binding domains 1 and 2 form stable molecular complexes. The in vitro co-localization of precursor microRNA 149 and nucleolin in PC3 cells was demonstrated using confocal microscopy. Finally, a rapid and straightforward microfluidic strategy was employed to check the ability of precursor microRNA 149 to capture nucleolin RNA-binding domains 1 and 2. The results revealed that precursor microRNA 149 can capture nucleolin RNA-binding domains 1 and 2 labeled with Fluorescein 5-isothiocyanate in a concentration-dependent manner, but PhenDC3 complexation seems to decrease the ability of precursor microRNA 149 to capture the protein. Overall, our results proved the formation of the G-quadruplex structure in the precursor microRNA 149 and the ability to recognize and detect nucleolin. This proof-of-concept study could open up a new framework for developing new strategies to design improved molecular receptors for capture and detection of nucleolin in complex biological samples. publishersversion published
Databáze: OpenAIRE
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