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G-Clamp Heterocycle Modification Containing Interstrand Photo-Cross-Linker to Capture Intracellular MicroRNA Targets
Weiguo Shen1, Yongkang Hou1, Yunpeng Yi1
1State Key Laboratory of Natural and Biomimetic Drugs, Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
Abstract:
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.
Insights
Researchers developed ARAGON, a novel chemical probe for capturing intracellular microRNA (miRNA) targets. This method efficiently identifies miRNA-mRNA interactions, revealing GAB1 as a miR-101 target and its role in promoting lung cancer cell apoptosis.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- MicroRNAs (miRNAs) are crucial regulators of gene expression at the post-transcriptional level.
- Identifying miRNA targets is vital for understanding miRNA function and regulatory mechanisms.
- Current methods struggle to detect weak miRNA-mRNA interactions, hindering miRNA research.
Purpose of the Study:
- To develop an unbiased chemical strategy for capturing intracellular miRNA targets.
- To provide proof-of-concept for a novel photo-cross-linking approach to detect miRNA-mRNA interactions.
- To identify novel miRNA targets and elucidate their roles in cellular processes and disease.
Main Methods:
- Development of an aryl-diazirine-G-clamp-modified-nucleoside (ARAGON) miRNA probe.
- Utilizing ARAGON for covalent capture of intracellular miRNA targets via photo-cross-linking.
- Validation of ARAGON probes by enriching known miRNA targets (miR-106a, miR-21, miR-101).
- Application of ARAGON to screen for unknown miRNA targets in various cell lines.
Main Results:
- ARAGON probes demonstrated accuracy and effective binding properties.
- Successful enrichment of known miRNA targets, validating the probe's efficacy.
- Identification of GAB1 as a novel target of miR-101 in H1299 lung cancer cells.
- Demonstration that miR-101-mediated silencing of GAB1 promotes apoptosis in lung cancer cells.
Conclusions:
- The ARAGON chemical probe is a powerful and versatile tool for global miRNA target screening.
- This method enhances the detection of transient miRNA-mRNA interactions.
- The findings reveal a novel oncogenic mechanism involving miR-101 and GAB1 in lung cancer, suggesting therapeutic potential.
