Wang Ke, Liu Siqian, Zhou Shuqi, Qileng Aori, Wang Dingyi, Liu Yingju, Chen Chunlai, Lei Chunyang, Nie Zhou
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University, Changsha, 410082, P. R. China.
State Key Laboratory of Membrane Biology, Beijing Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing, 100084, P. R. China.
Angew Chem Int Ed Engl. 2025 Jan 21;64(4):e202416671. doi: 10.1002/anie.202416671. Epub 2024 Nov 27.
Natural protein-protein communications, such as those between transcription factors (TFs) and RNA polymerases/ribosomes, underpin cell-free biosensing systems operating on the transcription/translation (TXTL) paradigm. However, their deployment in field analysis is hampered by the delayed response (hour-level) and the complex composition of in vitro TXTL systems. For this purpose, we present a de novo-designed ligand-responsive artificial protein-protein communication (LIRAC) by redefining the connection between TFs and non-interacting CRISPR/Cas enzymes. By rationally designing a chimeric DNA adaptor and precisely regulating its binding affinities to both proteins, LIRAC immediately transduces target-induced TF allostery into rapid CRISPR/Cas enzyme activation within a homogeneous system. Consequently, LIRAC obviates the need for RNA/protein biosynthesis inherent to conventional TXTL-based cell-free systems, substantially reducing reaction complexity and time (from hours to 10 minutes) with improved sensitivity and tunable dynamic range. Moreover, LIRAC exhibits excellent versatility and programmability for rapidly and sensitively detecting diverse contaminants, including antibiotics, heavy metal ions, and preservatives. It also enables the creation of a multi-protein communication-based tristate logic for the intelligent detection of multiple contaminants. Integrated with portable devices, LIRAC has been proven effective in the field analysis of environmental samples and personal care products, showcasing its potential for environmental and health monitoring.
天然的蛋白质-蛋白质通讯，例如转录因子（TFs）与RNA聚合酶/核糖体之间的通讯，是基于转录/翻译（TXTL）范式运行的无细胞生物传感系统的基础。然而，它们在现场分析中的应用受到延迟响应（小时级）和体外TXTL系统复杂组成的阻碍。为此，我们通过重新定义TFs与非相互作用的CRISPR/Cas酶之间的联系，提出了一种从头设计的配体响应人工蛋白质-蛋白质通讯（LIRAC）。通过合理设计嵌合DNA接头并精确调节其与两种蛋白质的结合亲和力，LIRAC可在均质系统中立即将靶标诱导的TF变构转化为快速的CRISPR/Cas酶激活。因此，LIRAC无需传统基于TXTL的无细胞系统固有的RNA/蛋白质生物合成，大大降低了反应复杂性和时间（从数小时降至10分钟），同时提高了灵敏度和可调动态范围。此外，LIRAC在快速灵敏地检测多种污染物（包括抗生素、重金属离子和防腐剂）方面表现出出色的通用性和可编程性。它还能够创建基于多蛋白质通讯的三态逻辑，用于智能检测多种污染物。与便携式设备集成后，LIRAC已被证明在环境样品和个人护理产品的现场分析中有效，展示了其在环境和健康监测方面的潜力。
