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To What Extent Do Fluorophores Bias the Biological Activity of Peptides? A Practical Approach Using Membrane-Active Peptides as Models

The characterization of biologically active peptides relies heavily on the study of their efficacy, toxicity, mechanism of action, cellular uptake, or intracellular location, using both in vitro and in vivo studies. These studies frequently depend on the use of fluorescence-based techniques. Since m...

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Autores principales: Cavaco, Marco, Pérez-Peinado, Clara, Valle, Javier, Silva, Rúben D. M., Correia, João D. G., Andreu, David, Castanho, Miguel A. R. B., Neves, Vera
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7509492/
https://www.ncbi.nlm.nih.gov/pubmed/33015016
http://dx.doi.org/10.3389/fbioe.2020.552035
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author Cavaco, Marco
Pérez-Peinado, Clara
Valle, Javier
Silva, Rúben D. M.
Correia, João D. G.
Andreu, David
Castanho, Miguel A. R. B.
Neves, Vera
author_facet Cavaco, Marco
Pérez-Peinado, Clara
Valle, Javier
Silva, Rúben D. M.
Correia, João D. G.
Andreu, David
Castanho, Miguel A. R. B.
Neves, Vera
author_sort Cavaco, Marco
collection PubMed
description The characterization of biologically active peptides relies heavily on the study of their efficacy, toxicity, mechanism of action, cellular uptake, or intracellular location, using both in vitro and in vivo studies. These studies frequently depend on the use of fluorescence-based techniques. Since most peptides are not intrinsically fluorescent, they are conjugated to a fluorophore. The conjugation may interfere with peptide properties, thus biasing the results. The selection of the most suitable fluorophore is highly relevant. Here, a comprehensive study with blood–brain barrier (BBB) peptide shuttles (PepH3 and PepNeg) and antimicrobial peptides (AMPs) (vCPP2319 and Ctn[15-34]), tested as anticancer peptides (ACPs), having different fluorophores, namely 5(6)-carboxyfluorescein (CF), rhodamine B (RhB), quasar 570 (Q570), or tide fluor 3 (TF3) attached is presented. The goal is the evaluation of the impact of the selected fluorophores on peptide performance, applying routinely used techniques to assess cytotoxicity/toxicity, secondary structure, BBB translocation, and cellular internalization. Our results show that some fluorophores significantly modulate peptide activity when compared with unlabeled peptides, being more noticeable in hydrophobic and charged fluorophores. This study highlights the need for a careful experimental design for fluorescently labeled molecules, such as peptides.
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spelling pubmed-75094922020-10-02 To What Extent Do Fluorophores Bias the Biological Activity of Peptides? A Practical Approach Using Membrane-Active Peptides as Models Cavaco, Marco Pérez-Peinado, Clara Valle, Javier Silva, Rúben D. M. Correia, João D. G. Andreu, David Castanho, Miguel A. R. B. Neves, Vera Front Bioeng Biotechnol Bioengineering and Biotechnology The characterization of biologically active peptides relies heavily on the study of their efficacy, toxicity, mechanism of action, cellular uptake, or intracellular location, using both in vitro and in vivo studies. These studies frequently depend on the use of fluorescence-based techniques. Since most peptides are not intrinsically fluorescent, they are conjugated to a fluorophore. The conjugation may interfere with peptide properties, thus biasing the results. The selection of the most suitable fluorophore is highly relevant. Here, a comprehensive study with blood–brain barrier (BBB) peptide shuttles (PepH3 and PepNeg) and antimicrobial peptides (AMPs) (vCPP2319 and Ctn[15-34]), tested as anticancer peptides (ACPs), having different fluorophores, namely 5(6)-carboxyfluorescein (CF), rhodamine B (RhB), quasar 570 (Q570), or tide fluor 3 (TF3) attached is presented. The goal is the evaluation of the impact of the selected fluorophores on peptide performance, applying routinely used techniques to assess cytotoxicity/toxicity, secondary structure, BBB translocation, and cellular internalization. Our results show that some fluorophores significantly modulate peptide activity when compared with unlabeled peptides, being more noticeable in hydrophobic and charged fluorophores. This study highlights the need for a careful experimental design for fluorescently labeled molecules, such as peptides. Frontiers Media S.A. 2020-09-09 /pmc/articles/PMC7509492/ /pubmed/33015016 http://dx.doi.org/10.3389/fbioe.2020.552035 Text en Copyright © 2020 Cavaco, Pérez-Peinado, Valle, Silva, Correia, Andreu, Castanho and Neves. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Cavaco, Marco
Pérez-Peinado, Clara
Valle, Javier
Silva, Rúben D. M.
Correia, João D. G.
Andreu, David
Castanho, Miguel A. R. B.
Neves, Vera
To What Extent Do Fluorophores Bias the Biological Activity of Peptides? A Practical Approach Using Membrane-Active Peptides as Models
title To What Extent Do Fluorophores Bias the Biological Activity of Peptides? A Practical Approach Using Membrane-Active Peptides as Models
title_full To What Extent Do Fluorophores Bias the Biological Activity of Peptides? A Practical Approach Using Membrane-Active Peptides as Models
title_fullStr To What Extent Do Fluorophores Bias the Biological Activity of Peptides? A Practical Approach Using Membrane-Active Peptides as Models
title_full_unstemmed To What Extent Do Fluorophores Bias the Biological Activity of Peptides? A Practical Approach Using Membrane-Active Peptides as Models
title_short To What Extent Do Fluorophores Bias the Biological Activity of Peptides? A Practical Approach Using Membrane-Active Peptides as Models
title_sort to what extent do fluorophores bias the biological activity of peptides? a practical approach using membrane-active peptides as models
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7509492/
https://www.ncbi.nlm.nih.gov/pubmed/33015016
http://dx.doi.org/10.3389/fbioe.2020.552035
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