生物正交化学特征使细胞氧化胸腺嘧啶的放大可视化,Analytical Chemistry

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生物正交化学特征使细胞氧化胸腺嘧啶的放大可视化
Analytical Chemistry ( IF 6.7 ) Pub Date : 2021-07-22 , DOI: 10.1021/acs.analchem.1c01285
Min Bai ₁ , Xiaowen Cao ₁ , Feng Chen ₁ , Jing Xue ₁ , Yue Zhao ₁ , Yongxi Zhao ₁

Affiliation

细胞氧化胸腺嘧啶、5-羟甲基尿嘧啶 (5hmU) 和 5-甲酰尿嘧啶 (5fU) 存在于多种生物的基因组中，其分布对生物过程和生命系统产生深远的影响。然而，由于缺乏用于单细胞分析的特定生物正交标记和灵敏度方法，尚未探索细胞氧化胸腺嘧啶的分布。在此，我们报告了一种生物正交化学特征，能够放大单个细胞中细胞氧化胸腺嘧啶的可视化。合成的 ATP-γ-炔烃，一种在 γ-磷酸上修饰生物正交标记的 ATP 类似物，可以通过化学酶标记与细胞 5hmU 特异性连接。然后用叠氮化物（N ₃)-修饰的 5hmU-引物。5fU 的鉴定基于从 5fU 到 5hmU 的选择性还原，随后对新生成的 5hmU 进行化学酶标记，并与 N ₃交联- 通过点击化学修饰的 5fU 引物。然后，所有 5hmU 和 5fU 位点都用各自的圆形条形码进行编码。这些条形码同时被放大以进行多路复用单分子成像。上述两种条码可同时放大，用于单细胞中5hmU和5fU的差异化可视化。我们发现这两种细胞氧化胸腺嘧啶在空间上以细胞类型依赖的方式组织，并具有细胞间异质性。我们还研究了它们的多级亚细胞信息，并探索了它们在细胞周期中的动态变化。此外，使用 DNA 测序代替荧光成像，

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Bioorthogonal Chemical Signature Enabling Amplified Visualization of Cellular Oxidative Thymines

Cellular oxidative thymines, 5-hydroxymethyluracil (5hmU) and 5-formyluracil (5fU), are found in the genomes of a diverse range of organisms, the distribution of which profoundly influence biological processes and living systems. However, the distribution of cellular oxidative thymines has not been explored because of lacking both specific bioorthogonal labeling and sensitivity methods for single-cell analysis. Herein, we report a bioorthogonal chemical signature enabling amplified visualization of cellular oxidative thymines in single cells. The synthesized ATP-γ-alkyne, an ATP analogue with bioorthogonal tag modified on γ-phosphate can be specifically linked to cellular 5hmU by chemoenzymatic labeling. DNA with 5-alkynephosphomethyluracil were then clicked with azide (N₃)-modified 5hmU-primer. Identification of 5fU is based on selective reduction from 5fU to 5hmU, subsequent chemoenzymatic labeling of the newly generated 5hmU, and cross-linking with N₃-modified 5fU-primer via click chemistry. Then, all of the 5hmU and 5fU sites are encoded with respective circularized barcodes. These barcodes are simultaneously amplified for multiplexed single-molecule imaging. The above two kinds of barcodes can be simultaneously amplified for differentiated visualization of 5hmU and 5fU in single cells. We find these two kinds of cellular oxidative thymines are spatially organized in a cell-type-dependent style with cell-to-cell heterogeneity. We also investigate their multilevel subcellular information and explore their dynamic changes during cell cycles. Further, using DNA sequencing instead of fluorescence imaging, our proposed bioorthogonal chemical signature holds great potential to offer the sequence information of these oxidative thymines in cells and may provide a reliable chemical biology approach for studying the whole-genome oxidative thymines profiles and insights into their functional role and dynamics in biology.

更新日期：2021-08-03

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