Table_1_Aptamer-Based Western Blot for Selective Protein Recognition.docx

Selective protein recognition is critical in molecular biology techniques such as Western blotting and ELISA. Successful detection of the target proteins in these methods relies on the specific interaction of the antibodies, which often bring a high production cost and require a long incubation time. Aptamers represent an alternative class of simple and affordable affinity reagents for protein recognition, and replacing antibodies with aptamers in Western blotting would potentially be more time- and cost-effective. In this work, multiple fluorescent DNA aptamers were isolated by in vitro selection to selectively label commonly used tag proteins including GST, MBP, and His-tag. The generated aptamers G1, M1, and H1 specifically bound to their cognate target proteins with nanomolar affinities, respectively. Compared with conventional antibody-based immunoblotting, such aptamer-based procedure gave a cleaner background and was able to selectively label target protein in a complex mixture. Lastly, the identified aptamers were also effective in recognition of different fusion proteins with the same tag, thus greatly expanding the scope of the potential applications of these aptamers. This work provided aptamers as useful molecular tools for selective protein recognition in Western blotting analysis.

选择性蛋白识别在蛋白质印迹法（Western blotting）、酶联免疫吸附测定（ELISA）等分子生物学技术中至关重要。此类方法中靶蛋白的成功检测依赖于抗体的特异性结合，但抗体往往存在制备成本高昂、孵育周期较长的缺陷。适配体（Aptamers）是一类简易且经济的亲和试剂，可作为抗体的替代方案用于蛋白识别；在蛋白质印迹法中用适配体取代抗体，有望在时间与成本上更具优势。本研究通过体外筛选技术分离得到多种荧光DNA适配体，可特异性标记常用的标签蛋白，包括谷胱甘肽S-转移酶标签（GST）、麦芽糖结合蛋白标签（MBP）与组氨酸标签（His-tag）。所获得的适配体G1、M1与H1分别对各自对应的靶蛋白展现出纳摩尔级的结合亲和力。与传统基于抗体的免疫印迹法相比，基于适配体的检测流程可获得更清晰的背景信号，且能够在复杂混合物中选择性标记靶蛋白。此外，所鉴定得到的适配体同样可有效识别携带同一标签的不同融合蛋白，极大拓展了这些适配体的潜在应用范围。本研究开发的适配体可作为实用的分子工具，用于蛋白质印迹分析中的选择性蛋白识别。