Department of Chemistry, Stanford University, Stanford, California 94305-5080, USA.
J Am Chem Soc. 2013 Apr 24;135(16):6184-91. doi: 10.1021/ja4004393. Epub 2013 Apr 16.
Genetically encoded methods for protein conjugation are of high importance as biological tools. Here we describe the development of a new class of dyes for genetically encoded tagging that add new capabilities for protein reporting and detection via HaloTag methodology. Oligodeoxyfluorosides (ODFs) are short DNA-like oligomers in which the natural nucleic acid bases are replaced by interacting fluorescent chromophores, yielding a broad range of emission colors using a single excitation wavelength. We describe the development of an alkyl halide dehalogenase-compatible chloroalkane linker phosphoramidite derivative that enables the rapid automated synthesis of many possible dyes for protein conjugation. Experiments to test the enzymatic self-conjugation of nine different DNA-like dyes to proteins with HaloTag domains in vitro were performed, and the data confirmed the rapid and efficient covalent labeling of the proteins. Notably, a number of the ODF dyes were found to increase in brightness or change color upon protein conjugation. Tests in mammalian cellular settings revealed that the dyes are functional in multiple cellular contexts, both on the cell surface and within the cytoplasm, allowing protein localization to be imaged in live cells by epifluorescence and laser confocal microscopy.
基因编码的蛋白质偶联方法是非常重要的生物工具。在这里，我们描述了一类新的染料的发展，用于基因编码标记，通过 HaloTag 方法为蛋白质报告和检测增加新的功能。寡脱氧氟化物（ODFs）是短的类似 DNA 的寡聚物，其中天然核酸碱基被相互作用的荧光生色团取代，使用单一激发波长产生广泛的发射颜色。我们描述了一种烷基卤化物脱卤酶相容的氯代烷烃连接体膦酰胺衍生物的开发，该衍生物能够快速自动化合成许多可能用于蛋白质偶联的染料。在体外进行了九种不同的类似 DNA 的染料与带有 HaloTag 结构域的蛋白质的酶促自偶联实验，数据证实了蛋白质的快速和有效的共价标记。值得注意的是，一些 ODF 染料在蛋白质偶联时亮度增加或颜色发生变化。在哺乳动物细胞环境中的测试表明，这些染料在多种细胞环境中都具有功能，包括在细胞表面和细胞质内，允许通过荧光和激光共聚焦显微镜对活细胞中的蛋白质定位进行成像。
