Guo Weijie, Ma Yuan, Mou Quanbing, Shao Xiangli, Lyu Mingkuan, Garcia Valeria, Kong Linggen, Lewis Whitney, Yang Zhenglin, Lu Shuya, Lu Yi
Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA.
Interdisciplinary Life Sciences Graduate Programs, The University of Texas at Austin, Austin, TX, USA.
Nat Protoc. 2025 Jan 8. doi: 10.1038/s41596-024-01103-x.
Glycosylated RNAs (glycoRNAs) have recently emerged as a new class of molecules of substantial interest owing to their potential roles in cellular processes and diseases. However, studying glycoRNAs is challenging owing to the lack of effective research tools including, but not limited to, imaging techniques to study the spatial distribution of glycoRNAs. Recently, we reported the development of a glycoRNA imaging technique, called sialic acid aptamer and RNA in situ hybridization-mediated proximity ligation assay (ARPLA), to visualize sialic acid-containing glycoRNAs with high sensitivity and specificity. Here we describe the experimental design principles and detailed step-by-step procedures for ARPLA-assisted glycoRNA imaging across multiple cell types. The procedure includes details for target selection, oligo design and preparation, optimized steps for RNA in situ hybridization, glycan recognition, proximity ligation, rolling circle amplification and a guideline for image acquisition and analysis. With properly designed probe sets and cells prepared, ARPLA-based glycoRNA imaging can typically be completed within 1 d by users with expertise in biochemistry and fluorescence microscopy. The ARPLA approach enables researchers to explore the spatial distribution, trafficking and functional contributions of glycoRNAs in various cellular processes.
糖基化RNA（glycoRNAs）最近作为一类具有重大研究价值的新分子出现，因为它们在细胞过程和疾病中具有潜在作用。然而，由于缺乏有效的研究工具，包括但不限于用于研究glycoRNAs空间分布的成像技术，研究glycoRNAs具有挑战性。最近，我们报道了一种名为唾液酸适配体与RNA原位杂交介导的邻近连接分析（ARPLA）的glycoRNA成像技术的开发，用于以高灵敏度和特异性可视化含唾液酸的glycoRNAs。在这里，我们描述了跨多种细胞类型进行ARPLA辅助glycoRNA成像的实验设计原则和详细的逐步程序。该程序包括靶标选择、寡核苷酸设计和制备的细节、RNA原位杂交的优化步骤、聚糖识别、邻近连接、滚环扩增以及图像采集和分析指南。通过正确设计的探针组和制备的细胞，具有生物化学和荧光显微镜专业知识的用户通常可以在1天内完成基于ARPLA的glycoRNA成像。ARPLA方法使研究人员能够探索glycoRNAs在各种细胞过程中的空间分布、运输和功能作用。
