Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, CA, USA.
Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA.
Nat Methods. 2020 Mar;17(3):311-318. doi: 10.1038/s41592-019-0726-y. Epub 2020 Feb 3.
Tissues and organs are composed of diverse cell types, which poses a major challenge for cell-type-specific profiling of gene expression. Current metabolic labeling methods rely on exogenous pyrimidine analogs that are only incorporated into RNA in cells expressing an exogenous enzyme. This approach assumes that off-target cells cannot incorporate these analogs. We disprove this assumption and identify and characterize the enzymatic pathways responsible for high background incorporation. We demonstrate that mammalian cells can incorporate uracil analogs and characterize the enzymatic pathways responsible for high background incorporation. To overcome these limitations, we developed a new small molecule-enzyme pair consisting of uridine/cytidine kinase 2 and 2'-azidouridine. We demonstrate that 2'-azidouridine is only incorporated in cells expressing uridine/cytidine kinase 2 and characterize selectivity mechanisms using molecular dynamics and X-ray crystallography. Furthermore, this pair can be used to purify and track RNA from specific cellular populations, making it ideal for high-resolution cell-specific RNA labeling. Overall, these results reveal new aspects of mammalian salvage pathways and serve as a new benchmark for designing, characterizing and evaluating methodologies for cell-specific labeling of biomolecules.
组织和器官由多种细胞类型组成，这对细胞类型特异性基因表达谱分析构成了重大挑战。目前的代谢标记方法依赖于外源性嘧啶类似物，这些类似物仅在表达外源性酶的细胞中掺入 RNA 中。这种方法假设非靶细胞不能掺入这些类似物。我们否定了这一假设，并确定和表征了负责高背景掺入的酶途径。我们证明哺乳动物细胞可以掺入尿嘧啶类似物，并表征了负责高背景掺入的酶途径。为了克服这些限制，我们开发了一种由尿苷/胞苷激酶 2 和 2'-叠氮尿嘧啶组成的新的小分子-酶对。我们证明只有在表达尿苷/胞苷激酶 2 的细胞中才会掺入 2'-叠氮尿嘧啶，并使用分子动力学和 X 射线晶体学来表征选择性机制。此外，该对可以用于从特定细胞群体中纯化和跟踪 RNA，使其成为高分辨率细胞特异性 RNA 标记的理想选择。总之，这些结果揭示了哺乳动物补救途径的新方面，并为设计、表征和评估生物分子细胞特异性标记的方法提供了新的基准。
