A Förster Resonance Energy Transfer‐Based Ratiometric Sensor with the Allosteric Transcription Factor TetR. Issue 17 (6th April 2020) - Nguyen, Thuy T.
Abstract: A recent description of an antibody‐free assay is significantly extended for small molecule analytes using allosteric transcription factors (aTFs) and Förster resonance energy transfer (FRET). The FRET signal indicates the differential binding of an aTF–DNA pair with a dose‐dependent response to its effector molecule, i.e., the analyte. The new sensors described here, based on the well‐characterized aTF TetR, demonstrate several new features of the assay approach: 1) the generalizability of the sensors to additional aTF–DNA–analyte systems, 2) sensitivity modulation through the choice of donor fluorophore (quantum dots or fluorescent proteins, FPs), and 3) sensor tuning using aTF variants with differing aTF–DNA binding affinities. While all of these modular sensors self‐assemble, the design reported here based on a recombinant aTF–FP chimera with commercially available dye‐labeled DNA uses readily accessible sensor components to facilitate easy adoption of the sensing approach by the broader community. Abstract : Allosteric transcription factor TetR acts as a sensor and an actuator in two Förster resonance energy transfer (FRET) sensor designs to quantify the small molecule anhydrotetracycline. The homogeneous assays use minimal components to produce ratiometric fluorescent readouts based on dose‐dependent analyte‐responsive protein–DNA binding. Component accessibility and ease of assembly facilitate adoption of this generalizable sensor design.