基于双功能纳米团簇探针的单细胞RNA剪接变异体分析研究

Differential expression of RNA splicing variants among individual cells accounts for cell heterogeneity of gene expression, which plays key role in the immune system. Analysis of RNA splicing variants at single-cell level is of great significance for studying gene expression regulation, cell heterogeneity, and mechanism of immune disease. However, now available techniques are difficult to achieve single-cell analysis of RNA splice variants with high base resolution, spatial resolution and accurate quantification. To address these challenges, this project will construct DNA templated dual-functional nanocluster probes to achieve in situ imaging and accurate quantification of RNA splicing variants at single-cell level. By designing probes to directly targeting the splicing junction sequence of RNA splicing variants, the base recognition resolution is significantly improved. By using the controllable fluorescence of nanoclusters, in situ imaging and typing of RNA splicing variants are achieved. Due to the atom-precise nanocluster, RNA splicing variants can be accurately quantified by laser ablation mass spectrometry at single-cell level. Furthermore, the probes are applied to analyze RNA splicing variants of mononuclear macrophage under immune activation, exploring RNA-splicing variant-mediated immune heterogeneity. This strategy will provide a new single-cell analysis tool for studying the functional diversity of immune cells and immune disease mechanisms.

细胞间RNA剪接变异体差异性是导致基因表达异质性的主要因素，其在免疫系统中发挥关键作用。单细胞层次分析RNA剪接变异体对研究基因表达调控、细胞异质行为及免疫疾病发生机制至关重要。然而目前难以实现对RNA剪接变异体的高碱基分辨、空间分辨和精准定量的单细胞分析。本项目拟构建以DNA为模板的双功能纳米团簇探针，以实现RNA剪接变异体的原位成像，并同时实现单细胞精准定量。通过设计直接靶向RNA剪接变异体拼接位点的探针，显著提高碱基识别分辨率。通过利用纳米团簇的可控性荧光实现RNA剪接变异体的细胞原位成像及分型，同时借助纳米团簇精确可控金属原子数的特点，结合激光消蚀质谱实现RNA剪接变异体的单细胞精准定量。并进一步应用于免疫状态下单核巨噬细胞RNA剪接变异体单细胞水平分析，探索RNA剪接变异体介导的免疫异质性，为研究免疫细胞功能多样性及免疫疾病机制提供了单细胞分析工具。

细胞间RNA剪接变异体的差异表达是导致基因表达异质性的主要因素，在免疫系统的调节中起着关键作用。然而，现有技术难以实现对RNA剪接变异体的高碱基分辨、空间分辨和精准定量的单细胞分析。我们开发了以DNA为模板的双功能纳米团簇探针，可在单细胞水平实现对RNA剪接变异体的原位成像及精准定量。（1）得益于纳米团簇探针空间位阻小和直接靶向RNA剪接拼接位点的特点，实现了RNA剪接变体的高碱基识别分辨率。（2）基于纳米团簇探针的可控荧光和不同金属元素类型，实现了RNA剪接变异体的原位成像和基因分型。（3）由于纳米团簇具有精确原子数的特点，通过激光剥蚀电感耦合等离子体质谱计数金或银原子，可在单细胞水平上准确定量RNA剪接变异体。我们进一步应用该探针探索了单核巨噬细胞中MyD88剪接变异体在免疫激活下的功能。研究鉴定出MyD88S和MyD88L在免疫刺激后表达水平均提高。此外，我们还发现，在LPS刺激下MyD88S和MyD88L的单细胞相关系数增加，这是以前基于细胞群体的技术所没有观察到的。该方法可用来研究细胞间的变异性，并有望破译免疫功能多样性和疾病背后的因果机制。