(182aw) High-Throughput Operator Engineering for Tunable Atf-Based Biosensors

Small molecule sensing plays a crucial role in environmental, health, and pharmaceutical monitoring, with a rapidly increasing demand for detecting toxins, antibiotics, molecular markers, drugs, and pollutants. Traditional laboratory-based techniques, such as liquid chromatography-mass spectrometry (LC-MS) and enzyme-linked immunosorbent assays (ELISA), have proven effective but are often limited by their reliance on intensive analyte-specific bio-recognition elements. In response to these limitations, allosteric transcription factors (aTFs)—naturally occurring proteins within bacterial biomes—have emerged as a versatile and promising alternative for small molecule sensing. aTFs consist of an effector binding domain (EBD) that senses effectors, triggering conformational changes that modulate their DNA-binding domain (DBD) affinity in a switch-like manner. Ligand binding events can either facilitate or impede RNA polymerase-promoter interactions, enabling transcriptional activation or repression.

Mathematical models reveal two critical equilibria—aTF-DNA binding (aTF+DNA) and aTF-effector binding (aTF+effector)—governing biosensor sensitivity. Adjusting the dissociation constant (K_d) between aTF and DNA or effector enables tuning of the half-maximal inhibitory concentration (I₅₀) and the maximal slope of the dissociation curve (k_max). However, engineering TF mutants with optimal K_d values typically requires complex protein modifications and labor-intensive screening. In contrast, operator sequence engineering offers a more streamlined approach by modifying the DNA sequences that TFs bind to, allowing for fine-tuning of sensitivity, specificity, and dynamic range without the complexities of protein engineering.

To systematically evaluate operator sequences, we repurposed protein binding microarrays (PBMs)—a high-throughput platform for in vitro characterization of DNA-TF interactions. Agilent microarrays were functionalized with operator variants containing 1–2 base pair mutations within two binding sites, generating symmetrical and asymmetrical dyads. Each microarray contained four identical chambers with immobilized DNA probes. ssDNA in triplicates of the forward/reverse orientations were double-stranded via on-chip PCR while fluorophore-conjugated antibodies detected epitope-tagged TFs bound to DNA.

DNA Sequence Conservation Analysis revealed several important takeaways. Position weight matrices (PWMs) and k-mer analysis of Chambers 1 (MphR) and 3 (TetR) identified conserved nucleotides and motifs critical for binding. Ladder plots comparing fluorescence intensity between TF-only (Chambers 1/3) and TF+ligand (Chambers 2/4) chambers revealed significant binding reduction upon ligand addition. Variants with weakened K_d values showed steeper dissociation slopes, potentially enabling tunable sensitivity. Deeper understanding of the binding affinities for the operator sequences that can be used in the aTF-DNA-effector molecule binding schemes will be used to extend the dynamic range of bead-based optical sensors for the rapid readouts of multiplexed analyte detection across wide concentration ranges.