title: Design Principles for SuCESsFul Biosensors: Specific Fluorophore/Analyte Binding and Minimization of Fluorophore/Scaffold Interactions.
abstract: 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.
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
authors.forename: Seymour
authors.initials: S
authors.lastname: de Picciotto
authors.affiliation: Department of Chemistry, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA.
authors.forename: Paige M
authors.initials: PM
authors.lastname: Dickson
authors.affiliation: Koch Institute for Integrative Cancer Research, 500 Main Street, Cambridge, MA 02139, USA.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Traxlmayr
authors.affiliation: Johnson Research Foundation and Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: Bryan S
authors.initials: BS
authors.lastname: Marques
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 01239, USA.
authors.forename: Elke
authors.initials: E
authors.lastname: Socher
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
authors.forename: Sixing
authors.initials: S
authors.lastname: Zhao
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 01239, USA.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Cheung
authors.affiliation: Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology, Zurich, 8093, Switzerland.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Kiefer
authors.affiliation: Johnson Research Foundation and Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: A Joshua
authors.initials: AJ
authors.lastname: Wand
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA; Johnson Research Foundation and Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: Linda G
authors.initials: LG
authors.lastname: Griffith
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 01239, USA; Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
authors.forename: Barbara
authors.initials: B
authors.lastname: Imperiali
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, 500 Main Street, Cambridge, MA 02139, USA; Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. Electronic address: wittrup@MIT.EDU.
authors.forename: K Dane
authors.initials: KD
authors.lastname: Wittrup
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D005453:Fluorescence; D011506:Proteins / Q000032:analysis*; D062211:Spatio-Temporal Analysis
chemical_list: D011506:Proteins
keywords: Sso7d scaffold; directed evolution; protein engineering; sensors; solvatochromism