Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA.
711th Human Performance Wing, Air Force Research Laboratory, Wright Patterson Air Force Base, OH, USA.
Nat Commun. 2024 Nov 19;15(1):10001. doi: 10.1038/s41467-024-54260-8.
Allosteric transcription factors (aTF) regulate gene expression through conformational changes induced by small molecule binding. Although widely used as biosensors, aTFs have proven challenging to design for detecting new molecules because mutation of ligand-binding residues often disrupts allostery. Here, we develop Sensor-seq, a high-throughput platform to design and identify aTF biosensors that bind to non-native ligands. We screen a library of 17,737 variants of the aTF TtgR, a regulator of a multidrug exporter, against six non-native ligands of diverse chemical structures - four derivatives of the cancer therapeutic tamoxifen, the antimalarial drug quinine, and the opiate analog naltrexone - as well as two native flavonoid ligands, naringenin and phloretin. Sensor-seq identifies biosensors for each of these ligands with high dynamic range and diverse specificity profiles. The structure of a naltrexone-bound design shows shape-complementary methionine-aromatic interactions driving ligand specificity. To demonstrate practical utility, we develop cell-free detection systems for naltrexone and quinine. Sensor-seq enables rapid and scalable design of new biosensors, overcoming constraints of natural biosensors.
变构转录因子（aTF）通过小分子结合诱导的构象变化来调节基因表达。虽然变构转录因子被广泛用作生物传感器，但由于配体结合残基的突变经常破坏变构作用，因此设计用于检测新分子的变构转录因子具有挑战性。在这里，我们开发了 Sensor-seq，这是一种高通量平台，用于设计和鉴定与非天然配体结合的 aTF 生物传感器。我们针对六种不同化学结构的非天然配体（四种癌症治疗药物他莫昔芬的衍生物、抗疟药奎宁和阿片类药物纳曲酮，以及两种天然类黄酮配体柚皮苷和根皮苷），筛选了 TtgR（一种多药外排泵调节剂）的 17737 种变体文库。TtgR 是一种变构转录因子。Sensor-seq 为这些配体中的每一种都鉴定出了具有高动态范围和不同特异性特征的生物传感器。一个与纳曲酮结合的设计的结构显示出形状互补的甲硫氨酸-芳香族相互作用，从而驱动配体特异性。为了证明实际用途，我们开发了纳曲酮和奎宁的无细胞检测系统。Sensor-seq 能够快速、大规模地设计新型生物传感器，克服了天然生物传感器的限制。
