Ren Xiaojun, Deng Ruijie, Zhang Kaixiang, Sun Yupeng, Teng Xucong, Li Jinghong
Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China.
School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing 100081, China.
ACS Cent Sci. 2018 Jun 27;4(6):680-687. doi: 10.1021/acscentsci.8b00081. Epub 2018 May 30.
Immune cell heterogeneity due to the differential expression of RNA splicing variants still remains unexplored. This is mainly because single-cell imaging technologies of splicing variants with precise sequence or base resolution are now not readily available. Herein, we design a splice-junction anchored padlock-probe-mediated rolling circle amplification assay (SpliceRCA) for single-cell imaging of splice isoforms of essential regulatory immune gene (CD45) upon T-cell activation. Two recognition regions in the padlock probe can target the splice-junction sequence, resulting in a close proximity for triggering one-target-one-amplicon amplification. With the read length of ∼30 nucleotides, this method allows discrimination of isoforms with single-base precision and quantification of isoforms with single-molecule resolution. We applied SpliceRCA to single-cell image splice variants of essential regulatory immune gene (CD45) upon T-cell activation. It is found that CD45RO isoform presents a distal nuclear spatial distribution and is coregulated with CD45RB upon activation. Our strategy provides a single-cell analysis platform to investigate the mechanism of complex immune responses and may further guide immunotherapy.
由于RNA剪接变体的差异表达导致的免疫细胞异质性仍未得到充分探索。这主要是因为目前还没有现成的具有精确序列或碱基分辨率的单细胞剪接变体成像技术。在此，我们设计了一种基于剪接连接锚定锁式探针介导的滚环扩增分析方法（SpliceRCA），用于在T细胞活化后对关键调节免疫基因（CD45）的剪接异构体进行单细胞成像。锁式探针中的两个识别区域可以靶向剪接连接序列，从而近距离触发单靶标单扩增子扩增。该方法的读取长度约为30个核苷酸，能够以单碱基精度区分异构体，并以单分子分辨率对异构体进行定量。我们将SpliceRCA应用于T细胞活化后关键调节免疫基因（CD45）的单细胞图像剪接变体分析。结果发现，CD45RO异构体呈现核远端空间分布，并且在活化时与CD45RB共同调节。我们的策略提供了一个单细胞分析平台，用于研究复杂免疫反应的机制，并可能进一步指导免疫治疗。
