Wang Tao, Yin Wang, AlShamaileh Hadi, Zhang Yumei, Tran Phuong Ha-Lien, Nguyen Tuong Ngoc-Gia, Li Yong, Chen Kuisheng, Sun Miaomiao, Hou Yingchun, Zhang Weihong, Zhao Qingxia, Chen Changying, Zhang Pei-Zhuo, Duan Wei
1 School of Nursing, Zhengzhou University, Zhengzhou, P.R. China.
2 School of Medicine and Centre for Molecular and Medical Research, Deakin University, Waurn Ponds, Australia.
Hum Gene Ther Methods. 2019 Feb;30(1):1-16. doi: 10.1089/hgtb.2018.237.
As a nucleic acid alternative to traditional antibody, aptamer holds great potential in various fields of biology and medicine such as targeted gene therapy, drug delivery, bio-sensing, and laboratory medicine. Over the past decades, the conventional Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method has undergone dramatic modifications and improvements owing to developments in material sciences and analytical techniques. However, many of the recently developed strategies either require complex materials and instruments or suffer from low efficiency and high failure rates in the selection of desired aptamers. Accordingly, the development of aptamers against new or novel targets is still a major obstacle for aptamer-based research and application. Here, an improved protein-SELEX procedure is presented for simplified and highly efficient isolation of aptamers against protein targets. Approaches are described that ensure a high success rate in aptamer selection by simplifying polymerase chain reaction procedures, introducing denature gel, utilizing an electro-elution-based single-stranded DNA separation strategy, as well as an enzyme-linked immunosorbent assay-based highly sensitive binding assay. In addition, a simplified sample preparation method for MiSeq-based next-generation sequencing is also introduced. While a recombinant protein as a bait protein for SELEX is discussed here, this protocol will also be invaluable for researchers wishing to develop aptamers against targets other than proteins such as small molecules, lipids, carbohydrates, cells, and micro-organisms for future gene therapy and/or diagnostics.
作为传统抗体的核酸替代物，适配体在靶向基因治疗、药物递送、生物传感和检验医学等生物学和医学的各个领域具有巨大潜力。在过去几十年中，由于材料科学和分析技术的发展，传统的指数富集配体系统进化（SELEX）方法经历了显著的改进。然而，许多最近开发的策略要么需要复杂的材料和仪器，要么在所需适配体的筛选中效率低下且失败率高。因此，针对新的或新颖靶标的适配体开发仍然是基于适配体的研究和应用的主要障碍。在此，提出了一种改进的蛋白质SELEX程序，用于简化和高效地分离针对蛋白质靶标的适配体。描述了通过简化聚合酶链反应程序、引入变性凝胶、利用基于电洗脱的单链DNA分离策略以及基于酶联免疫吸附测定的高度灵敏结合测定来确保适配体筛选成功率高的方法。此外，还介绍了一种基于MiSeq的下一代测序的简化样品制备方法。虽然本文讨论了作为SELEX诱饵蛋白的重组蛋白，但该方案对于希望开发针对除蛋白质以外的靶标（如小分子、脂质、碳水化合物、细胞和微生物）的适配体以用于未来基因治疗和/或诊断的研究人员也将非常有价值。
