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，这些类似物仅掺入到表达外源性酶的细胞中的 RNA 中。这种方法假定脱靶细胞不能掺入这些类似物。我们反驳了这一假设，并识别和表征了导致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，我们开发了一种新的小分子酶对，由尿苷/胞苷激酶 2 和 2'-叠氮尿苷组成。我们证明 2'-叠氮尿苷仅掺入表达尿苷/胞苷激酶 2 的细胞中，并使用分子动力学和 X 射线晶体学表征选择性机制。此外，这对可用于从特定细胞群中纯化和追踪 RNA，使其成为高分辨率细胞特异性 RNA 标记的理想选择。总体而言，这些结果揭示了哺乳动物补救途径的新方面，并作为设计、表征和评估生物分子细胞特异性标记方法的新基准。