Design Principles for SuCESsFul Biosensors: Specific Fluorophore/Analyte Binding and Minimization of Fluorophore/Scaffold Interactions
Author(s)
Dickson, Paige M.; Marques, Bryan S.; Zhao, Sixing; Kiefer, Jonathan D.; Wand, A. Joshua; Griffith, Linda G.; De Picciotto, Seymour; Traxlmayr, Michael; Socher, Elke Ricarda; Cheung, Stephanie; Imperiali, Barbara; Wittrup, Karl Dane; ... Show more Show less
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Quantifying protein location and concentration is critical for understanding function in situ. Scaffold conjugated to environment-sensitive fluorophore (SuCESsFul) biosensors, in which a reporting fluorophore is conjugated to a binding scaffold, can, in principle, detect analytes of interest with high temporal and spatial resolution. However, their adoption has been limited due to the extensive empirical screening required for their development. We sought to establish design principles for this class of biosensor by characterizing over 400 biosensors based on various protein analytes, binding proteins, and fluorophores. We found that the brightest readouts are attained when a specific binding pocket for the fluorophore is present on the analyte. Also, interaction of the fluorophore with the binding protein it is conjugated to can raise background fluorescence, considerably limiting sensor dynamic range. Exploiting these two concepts, we designed biosensors that attain a 100-fold increase in fluorescence upon binding to analyte, an order of magnitude improvement over the previously best-reported SuCESsFul biosensor. These design principles should facilitate the development of improved SuCESsFul biosensors. Keywords: solvatochromism; Sso7d scaffold; sensors; protein engineering; directed evolution
Date issued
2016-07Department
Massachusetts Institute of Technology. Department of Biological Engineering; Massachusetts Institute of Technology. Department of Biology; Massachusetts Institute of Technology. Department of Chemical Engineering; Massachusetts Institute of Technology. Department of Chemistry; Koch Institute for Integrative Cancer Research at MITJournal
Journal of Molecular Biology
Publisher
Elsevier BV
Citation
De Picciotto, Seymour et al. “Design Principles for SuCESsFul Biosensors: Specific Fluorophore/Analyte Binding and Minimization of Fluorophore/Scaffold Interactions.” Journal of Molecular Biology 428, 20 (October 2016): 4228–4241 © 2016 Elsevier Ltd
Version: Author's final manuscript
ISSN
0022-2836
1089-8638