Cavaco, MarcoPérez-Peinado, Clara, 1991-Valle, JavierSilva, Rúben D.M.Correia, João D.G.Andreu Martínez, DavidCastanho, Miguel A.R.B.Neves, Vera2020-10-072020-10-072020Cavaco M, Pérez-Peinado C, Valle J, Silva RDM, Correia JDG, Andreu D, Castanho MARB, Neves V. To what extent do fluorophores bias the biological activity of peptides? A practical approach using membrane-active peptides as models. Front Bioeng Biotechnol. 2020; 8:552035. DOI: 10.3389/fbioe.2020.5520352296-4185http://hdl.handle.net/10230/45409The 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.application/pdfeng© 2020 Cavaco, Pérez-Peinado, Valle, Silva, Correia, Andreu, Castanho and Neves. 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.info:eu-repo/semantics/articlehttp://dx.doi.org/10.3389/fbioe.2020.552035BBB peptide shuttlesAnticancer peptidesFluorescenceFluorophoreLabelinginfo:eu-repo/semantics/openAccess