Abstract
Compared with green fluorescent protein-based biosensors, red fluorescent protein (RFP)-based biosensors are inherently advantageous because of reduced phototoxicity, decreased autofluorescence and enhanced tissue penetration. However, existing RFP-based biosensors often suffer from small dynamic ranges, mislocalization and undesired photoconversion. In addition, the choice of available RFP-based biosensors is limited, and development of each biosensor requires substantial effort. Herein, we describe a general and convenient method, which introduces a genetically encoded noncanonical amino acid, 3-aminotyrosine, to the chromophores of green fluorescent protein-like proteins and biosensors for spontaneous and efficient green-to-red conversion. We demonstrated that this method could be used to quickly expand the repertoire of RFP-based biosensors. With little optimization, the 3-aminotyrosine-modified biosensors preserved the molecular brightness, dynamic range and responsiveness of their green fluorescent predecessors. We further applied spectrally resolved biosensors for multiplexed imaging of metabolic dynamics in pancreatic β-cells.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Archaeal Proteins / genetics
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Archaeal Proteins / metabolism
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Biosensing Techniques*
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Cell Line
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Cloning, Molecular
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Fluorescence Resonance Energy Transfer
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Gene Expression
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Genetic Vectors / chemistry
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Genetic Vectors / metabolism
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Glucose / pharmacology
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Green Fluorescent Proteins / analysis*
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Green Fluorescent Proteins / biosynthesis
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Green Fluorescent Proteins / genetics
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Humans
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Insulin-Secreting Cells / cytology
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Insulin-Secreting Cells / drug effects
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Insulin-Secreting Cells / metabolism
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Luminescent Proteins / analysis*
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Luminescent Proteins / biosynthesis
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Luminescent Proteins / genetics
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Methanocaldococcus / chemistry
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Methanocaldococcus / enzymology
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Mice
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Optical Imaging / methods*
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Protein Engineering / methods*
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Red Fluorescent Protein
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Tyrosine / analogs & derivatives*
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Tyrosine / genetics
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Tyrosine / metabolism
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Tyrosine-tRNA Ligase / genetics
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Tyrosine-tRNA Ligase / metabolism
Substances
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Archaeal Proteins
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Luminescent Proteins
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Recombinant Proteins
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Green Fluorescent Proteins
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3-aminotyrosine
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Tyrosine
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Tyrosine-tRNA Ligase
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Glucose