Chang Andrew L, McKeague Maureen, Liang Joe C, Smolke Christina D
Department of Chemistry, Stanford University , Stanford, CA 94305, United States.
Anal Chem. 2014 Apr 1;86(7):3273-8. doi: 10.1021/ac5001527. Epub 2014 Mar 14.
Nucleic acid aptamers function as versatile sensing and targeting agents for analytical, diagnostic, therapeutic, and gene-regulatory applications, but their limited characterization and functional validation have hindered their broader implementation. We report the development of a surface plasmon resonance-based platform for rapid characterization of kinetic and equilibrium binding properties of aptamers to small molecules. Our system is label-free and scalable and enables analysis of different aptamer-target pairs and binding conditions with the same platform. This method demonstrates improved sensitivity, flexibility, and stability compared to other aptamer characterization methods. We validated our assay against previously reported aptamer affinity and kinetic measurements and further characterized a diverse panel of 12 small molecule-binding RNA and DNA aptamers. We report the first kinetic characterization for six of these aptamers and affinity characterization of two others. This work is the first example of direct comparison of in vitro selected and natural aptamers using consistent characterization conditions, thus providing insight into the influence of environmental conditions on aptamer binding kinetics and affinities, indicating different possible regulatory strategies used by natural aptamers, and identifying potential in vitro selection strategies to improve resulting binding affinities.
核酸适配体可作为多功能的传感和靶向剂，用于分析、诊断、治疗及基因调控应用，但对它们的表征和功能验证有限，这阻碍了其更广泛的应用。我们报告了一种基于表面等离子体共振的平台的开发，用于快速表征适配体与小分子的动力学和平衡结合特性。我们的系统无需标记且可扩展，能够在同一平台上分析不同的适配体-靶标对及结合条件。与其他适配体表征方法相比，该方法具有更高的灵敏度、灵活性和稳定性。我们针对先前报道的适配体亲和力和动力学测量结果验证了我们的检测方法，并进一步表征了一组包含12种小分子结合RNA和DNA适配体的多样化样本。我们报告了其中6种适配体的首次动力学表征以及另外2种的亲和力表征。这项工作是首次在一致的表征条件下对体外筛选的适配体和天然适配体进行直接比较的实例，从而深入了解环境条件对适配体结合动力学和亲和力的影响，揭示天然适配体可能采用的不同调控策略，并确定潜在的体外筛选策略以提高所得的结合亲和力。
