Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, United States.
Methods Enzymol. 2023;682:413-428. doi: 10.1016/bs.mie.2022.08.053. Epub 2022 Dec 24.
In this chapter, a new approach to the selective modification of native proteins is discussed, using electrophilic covalent aptamers. These biochemical tools are generated through the site-specific incorporation of a label-transferring or crosslinking electrophile into a DNA aptamer. Covalent aptamers provide the ability to transfer a variety of functional handles to a protein of interest or to irreversibly crosslink to the target. Methods for the aptamer-mediated labeling and crosslinking of thrombin are described. Thrombin labeling is fast and selective, in both simple buffer and in human plasma and outcompetes nuclease-mediated degradation. This approach provides facile, sensitive detection of labeled protein by western blot, SDS-PAGE, and mass spectrometry.
在本章中，我们讨论了一种使用亲电共价适体对天然蛋白质进行选择性修饰的新方法。这些生化工具是通过在 DNA 适体中特异性引入标记转移或交联亲电试剂来生成的。共价适体提供了将各种功能基团转移到感兴趣的蛋白质上或不可逆地交联到靶标的能力。本文描述了用于凝血酶的适体介导标记和交联的方法。凝血酶标记快速且具有选择性，无论是在简单缓冲液中还是在人血浆中，都能竞争抑制核酸酶介导的降解。这种方法通过 Western blot、SDS-PAGE 和质谱法提供了对标记蛋白的简便、灵敏的检测。
