Computational generation and characterization of IsdA-binding aptamers with single-molecule FRET analysis,Biotechnology Journal

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Computational generation and characterization of IsdA-binding aptamers with single-molecule FRET analysis
Biotechnology Journal ( IF 3.2 ) Pub Date : 2023-08-18 , DOI: 10.1002/biot.202300076
Godfred O Sabbih ₁ , Kalani M Wijesinghe ₂ , Chamika Algama ₂ , Soma Dhakal ₂ , Michael K Danquah _{1,

3}

Affiliation

Staphylococcus aureus is a major foodborne bacterial pathogen. Early detection of S. aureus is crucial to prevent infections and ensure food quality. The iron-regulated surface determinant protein A (IsdA) of S. aureus is a unique surface protein necessary for sourcing vital iron from host cells for the survival and colonization of the bacteria. The function, structure, and location of the IsdA protein make it an important protein for biosensing applications relating to the pathogen. Here, we report an in-silico approach to develop and validate high-affinity binding aptamers for the IsdA protein detection using custom-designed in-silico tools and single-molecule Fluorescence Resonance Energy Transfer (smFRET) measurements. We utilized in-silico oligonucleotide screening methods and metadynamics-based methods to generate 10 aptamer candidates and characterized them based on the Dissociation Free Energy (DFE) of the IsdA-aptamer complexes. Three of the aptamer candidates were shortlisted for smFRET experimental analysis of binding properties. Limits of detection in the low picomolar range were observed for the aptamers, and the results correlated well with the DFE calculations, indicating the potential of the in-silico approach to support aptamer discovery.

中文翻译：

通过单分子 FRET 分析计算生成 IsdA 结合适体并对其进行表征

金黄色葡萄球菌是一种主要的食源性细菌病原体。早期检测金黄色葡萄球菌对于预防感染和确保食品质量至关重要。金黄色葡萄球菌的铁调节表面决定蛋白 A (IsdA) 是一种独特的表面蛋白，是从宿主细胞中获取细菌生存和定植所需的重要铁所必需的。 IsdA 蛋白的功能、结构和位置使其成为与病原体相关的生物传感应用的重要蛋白质。在这里，我们报告了一种计算机方法，使用定制设计的计算机工具和单分子荧光共振能量转移（smFRET）测量来开发和验证用于 IsdA 蛋白检测的高亲和力结合适体。我们利用计算机寡核苷酸筛选方法和基于元动力学的方法生成了 10 个候选适配体，并根据 IsdA-适配体复合物的解离自由能 (DFE) 对它们进行了表征。三个候选适体入围用于结合特性的 smFRET 实验分析。观察到适体在低皮摩尔范围内的检测限，结果与 DFE 计算良好相关，表明计算机方法支持适体发现的潜力。

更新日期：2023-08-18

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