A Förster Resonance Energy Transfer-Based Ratiometric Sensor with the Allosteric Transcription Factor TetR

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.

Keywords: Förster resonance energy transfer (FRET); antibody-free assays; biosensors; homogeneous assays; molecular recognition; quantum dots; small molecule quantification.

Figure 1.. Schematic representation of sensor mechanism.

An allosteric transcription factor (aTF) labeled with a donor fluorophore (red) binds its cognate DNA sequence labeled with an acceptor fluorophore (purple). In this bound state, the proximity of the fluorophores promotes Förster resonance energy transfer (FRET) from the donor to the acceptor following photoluminescent excitation of the donor fluorophore. The binding of the aTF effector ligand (aka, the molecular analyte) to the aTF induces a conformational change in the protein, reducing its binding affinity for the DNA. The release of the DNA from the aTF extends the distance between their respective fluorophores, reducing energy transfer, yielding an analyte dose-dependent change in FRET and the donor and acceptor fluorescence intensities. The aTF used here, TetR, is a homodimer with two ligand binding pockets per dimer.^[11]

Figure 2.. Relative donor photoluminescence (PL) intensity as a function of the amount of Cy5-labeled DNA acceptor for two sensor designs, shown schematically.

(A,B) TetR^C-tdTomato + Cy5-DNA (C,D) QD-TetR^C + Cy5-DNA. TetR^C is displayed in yellow in both A and C. In A, tdTomato is in red and an alpha helical linker with sequence AEAAAKEAAAKA is in blue. The bottom axis indicates the ratio of Cy5-DNA to TF (TetR^C), while the top axis indicates the ratio of Cy5-DNA to the donor fluorophore (tdTomato or QD) to account for the difference in the stoichiometry. The colored circles are data using the target binding sequence tetO; the solid lines are fits to a modified Hill equation. The scrambled sequence (grey) acts as a non-binding control for collisional quenching and non-specific binding; the dashed line exhibits the linear fit typical of Stern-Vollmer collisional quenching. Data are means ± standard deviations of n = 3.

Figure 3.. Sensor response to analyte titration.

Representative spectral data for the aTc dose-dependent change in photoluminescence intensity for the sensor comprising (A) tdTomato-TetR^C + Cy5-DNA or (B) QD-TetR^C + Cy5-DNA. Spectra are background subtracted to eliminate the effects of direct acceptor excitation. A selection of the analyte concentrations is plotted for visual clarity. Ratio of acceptor fluorescence intensity to donor fluorescence intensity as a function of aTc concentration for sensor comprising (C) tdTomato-TetR^C + Cy5-DNA or (D) QD-TetR^C + Cy5-DNA. The first sensor has a 1:1:3 ratio of tdTomato:TF:DNA, while the second has a 1:4:18 ratio of QD:TF:DNA. tdTomato (200 nM) and QD (50 nM) concentrations were selected to maintain a constant aTF concentration at 200 nM. Data are mean ± standard deviation of n=3.

Figure 4.. Comparison of ratiometric sensors for aTc.

(A) Normalized ratio of acceptor and donor fluorescence intensity (F_A/F_D) shows analyte concentration dependence. (B) Normalized linear ranges of sensor outputs indicate narrower linear ranges for the more sensitive probes.