Allosteric transcription factors (aTF), widely used as biosensors, have proven challenging to design for detecting novel molecules because mutation of ligand-binding residues often disrupts allostery. We developed Sensor-seq, a high-throughput platform to design and identify aTF biosensors that bind to non-native ligands. We screened 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 identified novel biosensors for each of these ligands with high dynamic range and diverse specificity profiles. The structure of a naltrexone-bound design showed shape-complementary methionine-aromatic interactions driving ligand specificity. To demonstrate practical utility, we developed cell-free detection systems for naltrexone and quinine. Sensor-seq enables rapid, scalable design of new biosensors, overcoming constraints of natural biosensors.

中文翻译：

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高度多重设计的变构转录因子来感知新型配体

广泛用作生物传感器的变构转录因子（aTF）已被证明设计用于检测新分子具有挑战性，因为配体结合残基的突变通常会破坏变构。我们开发了 Sensor-seq，这是一个高通量平台，用于设计和识别与非天然配体结合的 aTF 生物传感器。我们针对多种化学结构的六种非天然配体（癌症治疗药物他莫昔芬、抗疟药物奎宁和阿片类似物纳曲酮的四种衍生物）筛选了 aTF TtgR（多药输出蛋白的调节剂）的 17,737 个变体的库，结果如下：以及两种天然类黄酮配体，柚皮素和根皮素。 Sensor-seq 为这些配体中的每一种识别出具有高动态范围和不同特异性特征的新型生物传感器。纳曲酮结合设计的结构显示出驱动配体特异性的形状互补的蛋氨酸-芳香族相互作用。为了证明实用性，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、可扩展地设计新型生物传感器，克服天然生物传感器的限制。