Institute of Analytical Chemistry and Instrument for Life Science, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology , Xi'an Jiaotong University , Xianning West Road , Xi'an , Shaanxi 710049 , P. R. China.
Institute of Molecular Medicine, Renji Hospital, School of Medicine and School of Chemistry and ChemicalEngineering , Shanghai Jiao Tong University , Shanghai 200127 , P. R. China.
J Am Chem Soc. 2020 Feb 12;142(6):2889-2896. doi: 10.1021/jacs.9b11393. Epub 2020 Feb 4.
5-Hydroxymethyluracil ( 5hmU ) is found in the genomes of a diverse range of organisms as another kind of 5-hydroxymethylpyrimidine, with the exception of 5-hydroxymethylcytosine ( 5hmC ). The biological function of 5hmU has not been well explored due to lacking both specific 5hmU recognition and single-cell analysis methods. Here we report differentiated visualization of single-cell 5hmU and 5hmC with microfluidic hydrogel encoding (sc 5hmU / 5hmC -microgel). Single cells and their genomic DNA after cell lysis can be encapsulated in individual agarose microgels. The 5hmU sites are then specifically labeled with thiophosphate for the first time, followed by labeling 5hmC with azide glucose. These labeled bases are each encoded into respective DNA barcode primers by chemical cross-linking. In situ amplification is triggered for single-molecule fluorescence visualization of single-cell 5hmU and 5hmC . On the basis of the sc 5hmU / 5hmC -microgel, we reveal cell type-specific molecular signatures of these two bases with remarkable single-cell heterogeneity. Utilizing machine learning algorithms to decode four-dimensional signatures of 5hmU / 5hmC , we visualize the discrimination of nontumorigenic, carcinoma and highly invasive breast cell lines. This strategy provides a new route to analyze and decode single-cell DNA epigenetic modifications.
5-羟甲基尿嘧啶（5hmU）是除 5-羟甲基胞嘧啶（5hmC）外，在多种生物的基因组中发现的另一种 5-羟甲基嘧啶。由于缺乏特异性的 5hmU 识别和单细胞分析方法，5hmU 的生物学功能尚未得到充分探索。在这里，我们报告了使用微流控水凝胶编码（sc5hmU/5hmC-microgel）对单细胞 5hmU 和 5hmC 的差异化可视化。单细胞及其裂解后的基因组 DNA 可以封装在单个琼脂糖微凝胶中。然后，5hmU 位点首次被硫代磷酸酯特异性标记，然后用叠氮葡萄糖标记 5hmC。这些标记的碱基通过化学交联各自编码成相应的 DNA 条形码引物。原位扩增被触发，用于单细胞 5hmU 和 5hmC 的单分子荧光可视化。基于 sc5hmU/5hmC-microgel，我们揭示了这两种碱基的细胞类型特异性分子特征，具有显著的单细胞异质性。利用机器学习算法对 5hmU/5hmC 的四维特征进行解码，我们可视化了非致瘤性、癌性和高侵袭性乳腺细胞系的区分。该策略为分析和解码单细胞 DNA 表观遗传修饰提供了新途径。
