TY - JOUR
T1 - Membrane-permeant, environment-sensitive dyes generate biosensors within living cells
AU - Macnevin, Christopher J.
AU - Watanabe, Takashi
AU - Weitzman, Matthew
AU - Gulyani, Akash
AU - Fuehrer, Sheryl
AU - Pinkin, Nicholas K.
AU - Tian, Xu
AU - Liu, Feng
AU - Jin, Jian
AU - Hahn, Klaus M.
N1 - Publisher Copyright:
© 2019 American Chemical Society.
PY - 2019/4/16
Y1 - 2019/4/16
N2 - Dyes with environment-sensitive fluorescence have proven useful to study the spatiotemporal dynamics of protein activity in living cells. When attached to proteins, their fluorescence can reflect protein conformational changes, post-translational modifications, or protein interactions. However, the utility of such dye-protein conjugates has been limited because it is difficult to load them into cells. They usually must be introduced using techniques that perturb cell physiology, limit throughput, or generate fluorescent vesicles (e.g., electroporation, microinjection, or membrane transduction peptides). Here we circumvent these problems by modifying a proven, environment-sensitive biosensor fluorophore so that it can pass through cell membranes without staining intracellular compartments and can be attached to proteins within living cells using unnatural amino acid (UAA) mutagenesis. Reactive groups were incorporated for attachment to UAAs or small molecules (mero166, azide; mero167, alkyne; mero76, carboxylic acid). These dyes are bright and fluoresce at long wavelengths (reaching ϵ = 100 000 M-1 cm-1, φ = 0.24, with excitation 565 nm and emission 594 nm). The utility of mero166 was demonstrated by in-cell labeling of a UAA to generate a biosensor for the small GTPase Cdc42. In addition, conjugation of mero166 to a small molecule produced a membrane-permeable probe that reported the localization of the DNA methyltransferase G9a in cells. This approach provides a strategy to access biosensors for many targets and to more practically harness the varied environmental sensitivities of synthetic dyes.
AB - Dyes with environment-sensitive fluorescence have proven useful to study the spatiotemporal dynamics of protein activity in living cells. When attached to proteins, their fluorescence can reflect protein conformational changes, post-translational modifications, or protein interactions. However, the utility of such dye-protein conjugates has been limited because it is difficult to load them into cells. They usually must be introduced using techniques that perturb cell physiology, limit throughput, or generate fluorescent vesicles (e.g., electroporation, microinjection, or membrane transduction peptides). Here we circumvent these problems by modifying a proven, environment-sensitive biosensor fluorophore so that it can pass through cell membranes without staining intracellular compartments and can be attached to proteins within living cells using unnatural amino acid (UAA) mutagenesis. Reactive groups were incorporated for attachment to UAAs or small molecules (mero166, azide; mero167, alkyne; mero76, carboxylic acid). These dyes are bright and fluoresce at long wavelengths (reaching ϵ = 100 000 M-1 cm-1, φ = 0.24, with excitation 565 nm and emission 594 nm). The utility of mero166 was demonstrated by in-cell labeling of a UAA to generate a biosensor for the small GTPase Cdc42. In addition, conjugation of mero166 to a small molecule produced a membrane-permeable probe that reported the localization of the DNA methyltransferase G9a in cells. This approach provides a strategy to access biosensors for many targets and to more practically harness the varied environmental sensitivities of synthetic dyes.
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U2 - 10.1021/jacs.8b09841
DO - 10.1021/jacs.8b09841
M3 - Article
C2 - 30994345
AN - SCOPUS:85065441495
SN - 0002-7863
VL - 141
SP - 7275
EP - 7282
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 18
ER -