Evaluation of carboxyfluorescein-labeled 7-methylguanine nucleotides as probes for studying cap-binding proteins by fluorescence anisotropy

Abstract Fluorescence anisotropy (FA) is a powerful technique for the discovery of protein inhibitors in a high-throughput manner. In this study, we sought to develop new universal FA-based assays for the evaluation of compounds targeting mRNA 5′ cap-binding proteins of therapeutic interest, includi...

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Autores principales:	Anna Wojtczak, Renata Kasprzyk, Marcin Warmiński, Krystian Ubych, Dorota Kubacka, Pawel J. Sikorski, Jacek Jemielity, Joanna Kowalska
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/17b379a52296481f8cd837d1cb2a8f0d
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spelling	oai:doaj.org-article:17b379a52296481f8cd837d1cb2a8f0d2021-12-02T14:26:51ZEvaluation of carboxyfluorescein-labeled 7-methylguanine nucleotides as probes for studying cap-binding proteins by fluorescence anisotropy10.1038/s41598-021-87306-82045-2322https://doaj.org/article/17b379a52296481f8cd837d1cb2a8f0d2021-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-87306-8https://doaj.org/toc/2045-2322Abstract Fluorescence anisotropy (FA) is a powerful technique for the discovery of protein inhibitors in a high-throughput manner. In this study, we sought to develop new universal FA-based assays for the evaluation of compounds targeting mRNA 5′ cap-binding proteins of therapeutic interest, including eukaryotic translation initiation factor 4E and scavenger decapping enzyme. For this purpose, a library of 19 carboxyfluorescein probes based on 7-methylguanine nucleotides was evaluated as FA probes for these proteins. Optimal probe:protein systems were further investigated in competitive binding experiments and adapted for high-throughput screening. Using a small in-house library of compounds, we verified and confirmed the accuracy of the developed FA assay to study cap-binding protein binders. The applications of the most promising probes were then extended to include evaluation of allosteric inhibitors as well as RNA ligands. From this analysis, we confirmed the utility of the method to study small molecule ligands and evaluate differently 5′ capped RNAs.Anna WojtczakRenata KasprzykMarcin WarmińskiKrystian UbychDorota KubackaPawel J. SikorskiJacek JemielityJoanna KowalskaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-15 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Anna Wojtczak Renata Kasprzyk Marcin Warmiński Krystian Ubych Dorota Kubacka Pawel J. Sikorski Jacek Jemielity Joanna Kowalska Evaluation of carboxyfluorescein-labeled 7-methylguanine nucleotides as probes for studying cap-binding proteins by fluorescence anisotropy
description	Abstract Fluorescence anisotropy (FA) is a powerful technique for the discovery of protein inhibitors in a high-throughput manner. In this study, we sought to develop new universal FA-based assays for the evaluation of compounds targeting mRNA 5′ cap-binding proteins of therapeutic interest, including eukaryotic translation initiation factor 4E and scavenger decapping enzyme. For this purpose, a library of 19 carboxyfluorescein probes based on 7-methylguanine nucleotides was evaluated as FA probes for these proteins. Optimal probe:protein systems were further investigated in competitive binding experiments and adapted for high-throughput screening. Using a small in-house library of compounds, we verified and confirmed the accuracy of the developed FA assay to study cap-binding protein binders. The applications of the most promising probes were then extended to include evaluation of allosteric inhibitors as well as RNA ligands. From this analysis, we confirmed the utility of the method to study small molecule ligands and evaluate differently 5′ capped RNAs.
format	article
author	Anna Wojtczak Renata Kasprzyk Marcin Warmiński Krystian Ubych Dorota Kubacka Pawel J. Sikorski Jacek Jemielity Joanna Kowalska
author_facet	Anna Wojtczak Renata Kasprzyk Marcin Warmiński Krystian Ubych Dorota Kubacka Pawel J. Sikorski Jacek Jemielity Joanna Kowalska
author_sort	Anna Wojtczak
title	Evaluation of carboxyfluorescein-labeled 7-methylguanine nucleotides as probes for studying cap-binding proteins by fluorescence anisotropy
title_short	Evaluation of carboxyfluorescein-labeled 7-methylguanine nucleotides as probes for studying cap-binding proteins by fluorescence anisotropy
title_full	Evaluation of carboxyfluorescein-labeled 7-methylguanine nucleotides as probes for studying cap-binding proteins by fluorescence anisotropy
title_fullStr	Evaluation of carboxyfluorescein-labeled 7-methylguanine nucleotides as probes for studying cap-binding proteins by fluorescence anisotropy
title_full_unstemmed	Evaluation of carboxyfluorescein-labeled 7-methylguanine nucleotides as probes for studying cap-binding proteins by fluorescence anisotropy
title_sort	evaluation of carboxyfluorescein-labeled 7-methylguanine nucleotides as probes for studying cap-binding proteins by fluorescence anisotropy
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/17b379a52296481f8cd837d1cb2a8f0d
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Evaluation of carboxyfluorescein-labeled 7-methylguanine nucleotides as probes for studying cap-binding proteins by fluorescence anisotropy

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