A Generalizable Fluorescence Sensor Platform for Sample Preparation-Free Protein Detection.

Modern molecular detection assays such as enzyme-linked immunosorbent assays (ELISAs) offer excellent sensitivity and specificity, but typically require multiple reagents and extensive sample preparation, limiting their usefulness as rapid diagnostics. A generalizable biosensor platform is introduced that enables single-step, sample preparation-free detection of protein analytes with high sensitivity in complex samples. The NanoFluor system employs Janelia Fluor dyes coupled to a nanobody via HaloTag conjugation with a flexible glycine-serine linker, where the dye undergoes a switch from a non-fluorescent to a fluorescent state when the coupled nanobody binds to its target.

Synthetic design of farnesyl-electrostatic peptides for development of a protein kinase A membrane translocation switch.

Molecular switches that respond to a biochemical stimulus in cells have proven utility as a foundation for developing molecular sensors and actuators that could be used to address important biological questions. Developing a molecular switch unfortunately remains difficult as it requires elaborate coordination of sensing and actuation mechanisms built into a single molecule. Here, we rationally designed a molecular switch that changes its subcellular localization in response to an intended stimulus such as an activator of protein kinase A (PKA).

Rational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter.

Protein Kinase A (PKA) exists as a tetrameric holoenzyme which activates with increase of cAMP and plays an important role in many physiological processes including cardiac physiology, neuronal development, and adipocyte function. Although this kinase has been the subject of numerous biosensor designs, a single-fluorophore reporter that performs comparably to Förster resonance energy transfer (FRET) has not yet been reported. Here, we have used basic observations of electrostatic interactions in PKA substrate recognition mechanism and nucleus localization sequence motif to design a phosphorylation switch that shuttles between the cytosol and the nucleus, a strategy that should be generalizable to all basophilic kinases.