美国加州大学欧文分校Robert C. Spitale研究团队近日取得一项新成果。他们利用优化的化学遗传方法完成了细胞特异性RNA代谢的标记。 这一研究成果于2020年2月3日在线发表在国际学术期刊《自然—方法学》上

研究人员反对这一假设，并鉴别和确定了负责高背景掺入的酶促途径。研究人员证明哺乳动物细胞可以利用尿嘧啶类似物，并确定了负责高背景掺入的酶促途径。为了克服这些限制，研究人员开发了一种新的小分子-酶对，该酶对由尿苷/胞苷激酶2和2'-叠氮胍组成。研究证明2'-叠氮胍仅进入表达尿苷/胞苷激酶2的细胞中，并使用分子动力学和X射线晶体学揭示了其选择性机制。

此外，该酶对可用于纯化和跟踪特定细胞群中的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，并为设计、表征和评估生物分子的细胞特异性标记方法提供了新的基准。

据了解，组织和器官由多种细胞类型组成，这对基因表达的细胞类型特异性分析构成了重大挑战。当前的代谢标记方法依赖于外源嘧啶类似物，其仅在表达外源酶的细胞中标记RNA。该方法假定脱靶细胞不能掺入这些类似物。

附：英文原文

Title: An optimized chemical-genetic method for cell-specific metabolic labeling of RNA

Author: Sarah Nainar, Bonnie J. Cuthbert, Nathan M. Lim, Whitney E. England, Ke Ke, Kanika Sophal, Robert Quechol, David L. Mobley, Celia W. Goulding, Robert C. Spitale

Issue&Volume: 2020-02-03

Abstract: 

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.

DOI: 10.1038/s41592-019-0726-y

Source: https://www.nature.com/articles/s41592-019-0726-y