Membrane-Permeant, Environment-Sensitive Dyes Generate Biosensors within Living Cells

Abstract

Dyes with environment-sensitive fluorescence have proven useful to study the spatio-temporal dynamics of protein activity in living cells. When attached to proteins, their fluorescence can reflect protein conformational changes, posttranslational 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 an 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.

Design of Turn‐On Near‐Infrared Fluorescent Probes for Highly Sensitive and Selective Monitoring of Biopolymers

“…Furthermore, the traditional approach typically yields low S/B ratios for biopolymer detection due to high fluorescent background of an unbound probe. Thus, increasing S/B ratio commonly requires additional step(s), such as washing to remove unbound probes or a reliance on multistep bioconjugation protocols With the intent of making the simple single‐step detection of biopolymers possible, we report near‐infrared (NIR) turn‐on fluorescent sensing systems for selective and highly sensitive detection of protein targets.…”

Nanoreactors for Chemical Synthesis and Biomedical Applications

“…Very recently, MacNevin and co‐workers proposed a membrane‐permeant dye with environment‐sensitive fluorescence for intracellular imaging . After modifying with appropriate side chains, this fluorophore can easily penetrate cell membrane without any interference to cellular life activity.…”

Visualizing semipermeability of the cell membrane using a pH-responsive ratiometric AIEgen

“…15 Thanks to the enthusiastic endeavors made by scientists, a lot of uorescent bioprobes have been developed for various applications. [16][17][18][19] Drugs with inherent uorescence make real-time in situ tracking of drug molecules in vivo or in vitro possible, which is of critical importance in pharmaceutical research. However, so far, uorescent drugs as probes for monitoring ion trapping have scarcely been reported in spite of their signicance in studying drug delivery and drug distribution in the body.…”