Cui Susu, Wang Fan, Yang Weiwei, Yu Yongsheng, Li Yingfu
State Key Laboratory of Space Power-Sources, School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150001, China.
MIIT Key Laboratory of Critical Materials Technology for New Energy Conversion and Storage, Harbin Institute of Technology, Harbin 150001, China.
Anal Chem. 2024 Sep 12. doi: 10.1021/acs.analchem.4c03316.
DNA-templated reactions have found wide applications in sensing and drug discovery. However, this strategy has been limited to the use of nucleic acids as templating elements to direct the proximity effect. Herein, we describe a versatile rotein-emplated lit ptamer lick igation eaction (PT-SpA-CLR) in which the protein template-induced covalent proximity ligation of split aptamer elements enables translating protein/aptamer binding events into the output of ligated DNA products. A ligation yield of >80% is observed for three model protein templates, including VEGF, PDGF-BB, and SARS-CoV-2 S1. The ligation reaction compensates for the weakness of reduced binding affinity resulting from splitting the aptamer, as evidenced by an approximately 2-fold lower dissociation constant than the non-ligated system. This newly developed PT-SpA-CLR strategy is further integrated with colorimetric or fluorescent reporting mechanisms to achieve easy-to-use and low-cost biosensors utilizing ligation to produce a fully active G-quadruplex or an RNA-cleaving DNAzyme to report protein binding. Both assays can achieve specific detection of an intended protein target with a limit of detection at the picomolar level even when challenged in biological samples. The combined PT-SpA-CLR and versatile sensing strategies offer attractive universal platforms for efficient detection of protein biomarkers.
DNA模板反应已在传感和药物发现中得到广泛应用。然而，这种策略仅限于使用核酸作为模板元件来引导邻近效应。在此，我们描述了一种通用的蛋白质模板化分裂适体连接反应（PT-SpA-CLR），其中蛋白质模板诱导的分裂适体元件的共价邻近连接能够将蛋白质/适体结合事件转化为连接的DNA产物的输出。对于三种模型蛋白质模板，包括血管内皮生长因子（VEGF）、血小板衍生生长因子-BB（PDGF-BB）和严重急性呼吸综合征冠状病毒2 S1（SARS-CoV-2 S1），观察到连接产率>80%。连接反应弥补了因分裂适体而导致的结合亲和力降低的弱点，这通过解离常数比未连接系统低约2倍得到证明。这种新开发的PT-SpA-CLR策略进一步与比色或荧光报告机制相结合，以实现易于使用且低成本的生物传感器，利用连接产生完全活性的G-四链体或切割RNA的脱氧核酶来报告蛋白质结合。即使在生物样品中受到挑战，两种检测方法都能实现对目标蛋白质的特异性检测，检测限达到皮摩尔水平。PT-SpA-CLR与通用传感策略相结合，为高效检测蛋白质生物标志物提供了有吸引力的通用平台。
