Protein-Templated Click Ligation Reaction Triggered by Protein-Split Aptamer Interactions,Analytical Chemistry

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Protein-Templated Click Ligation Reaction Triggered by Protein-Split Aptamer Interactions
Analytical Chemistry ( IF 6.7 ) Pub Date : 2024-09-12 , DOI: 10.1021/acs.analchem.4c03316
Susu Cui ₁ , Fan Wang ₁ , Weiwei Yang _{1,

2} , Yongsheng Yu _{1,

2} , Yingfu Li ₃

Affiliation

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 protein-templated split aptamer click ligation reaction (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 VEGF₁₆₅, 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.

中文翻译：

蛋白质分裂适体相互作用引发的蛋白质模板点击连接反应

DNA 模板反应在传感和药物发现领域有着广泛的应用。然而，该策略仅限于使用核酸作为模板元件来指导邻近效应。在此，我们描述了一种通用的蛋白质模板分割适体点击连接反应（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DNA产物的输出。对于 VEGF ₁₆₅ 、 PDGF-BB 和 SARS-CoV-2 S1 等三种模型蛋白模板，观察到连接率为 >80%。连接反应补偿了因适体分裂而导致的结合亲和力降低的弱点，解离常数比非连接系统低约2倍就证明了这一点。这种新开发的 PT-SpA-CLR 策略进一步与比色或荧光报告机制集成，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-四联体或 RNA 切割 DNAzyme 来报告蛋白质结合。即使在生物样品中受到挑战，两种检测方法都可以实现对预期蛋白质靶标的特异性检测，检测限在皮摩尔水平。组合的 PT-SpA-CLR 和多功能传感策略为有效检测蛋白质生物标志物提供了有吸引力的通用平台。

更新日期：2024-09-12

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