MicroRNAs (miRNAs) play indispensable roles in post-transcriptional gene regulation. The identification of target mRNAs is essential for dissecting the recognition basis, dynamics, and regulatory mechanism of miRNA–mRNA interactions. However, the lack of an unbiased method for detecting weak miRNA–mRNA interactions remains a long-standing obstacle for miRNA research. Here, we develop and provide proof-of-concept evidence demonstrating a chemical G-clamp-enhanced photo-cross-linking strategy for covalent capture of intracellular miRNA targets in different cell lines. This approach relies on an aryl-diazirine-G-clamp-modified-nucleoside (ARAGON) miRNA probe containing an alkynyl group that improves the thermal stability of miRNA–target mRNA duplex molecules and can rapidly cross-link with the complementary strand upon UV 365 nm activation, enhancing the transient capture of mRNA targets. After validating the accuracy and binding properties of ARAGON-based miRNA probes through the successful enrichment for the known targets of miR-106a, miR-21, and miR-101, we then extend ARAGON’s application to screen for previously unknown targets of different miRNAs in various cell lines. Ultimately, results in this study uncover GAB1 as a target of miR-101 in H1299 lung cancer cells and show that miR-101 silencing of GAB1 can promote apoptosis in H1299 cells, suggesting an oncogenic mechanism of GAB1. This study thus provides a powerful and versatile tool for enhanced screening of global miRNA targets in cells to facilitate investigations of miRNA functions in fundamental cellular processes and disease pathogenesis.

中文翻译：

---

含有链间光交联剂的 G-Clamp 杂环修饰可捕获细胞内 MicroRNA 靶标

MicroRNA (miRNA) 在转录后基因调控中发挥着不可或缺的作用。目标 mRNA 的识别对于剖析 miRNA-mRNA 相互作用的识别基础、动力学和调控机制至关重要。然而，缺乏一种公正的方法来检测弱 miRNA-mRNA 相互作用仍然是 miRNA 研究的长期障碍。在这里，我们开发并提供概念验证证据，证明化学 G 夹增强光交联策略可共价捕获不同细胞系中的细胞内 miRNA 靶标。该方法依赖于含有炔基的芳基-二a zirine- G -clamp-修饰-核苷( ARAGON) miRNA 探针，该探针可提高 miRNA-靶标 mRNA 双链体分子的热稳定性，并可快速交联在 UV 365 nm 激活时与互补链结合，增强 mRNA 靶标的瞬时捕获。通过成功富集 miR-106a、miR-21 和 miR-101 的已知靶标，验证基于 ARAGON 的 miRNA 探针的准确性和结合特性后，我们将 ARAGON 的应用扩展到筛选不同 miRNA 的先前未知靶标。各种细胞系。最终，这项研究的结果揭示了 GAB1 作为 H1299 肺癌细胞中 miR-101 的靶标，并表明 miR-101 沉默 GAB1 可以促进 H1299 细胞凋亡，提示 GAB1 的致癌机制。因此，这项研究提供了一个强大且多功能的工具，用于加强细胞中全局 miRNA 靶标的筛选，以促进对基本细胞过程和疾病发病机制中 miRNA 功能的研究。