基于邻近杂交后引物交换扩增(m6A-PHPEA)的 m6A 甲基化的位点特异性多重原位成像。

Multiplexed In Situ Imaging of Site-Specific m6A Methylation with Proximity Hybridization Followed by Primer Exchange Amplification (m6A-PHPEA).

机构信息

The Key Laboratory of Zhejiang Province for Aptamers and Theranostics, Zhejiang Cancer Hospital, Hangzhou Institute of Medicine (HIM), Chinese Academy of Sciences, Hangzhou, Zhejiang 310022, P. R. China.

Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, Zhejiang 310024, P. R. China.

出版信息

ACS Nano. 2024 Oct 8;18(40):27537-27546. doi: 10.1021/acsnano.4c08407. Epub 2024 Sep 27.

Abstract

Post-transcriptional modification of N6-methyladenosine (m6A) is crucial for ribonucleic acid (RNA) metabolism and cellular function. The ability to visualize site-specific m6A methylation at the single-cell level would markedly enhance our understanding of its pivotal regulatory functions in the field of epitranscriptomics. Despite this, current in situ imaging techniques for site-specific m6A are constrained, posing a significant barrier to epitranscriptomic studies and pathological diagnostics. Capitalizing on the precise targeting capability of deoxyribonucleic acid (DNA) hybridization and the high specificity of the m6A antibody, we present a method, termed proximity hybridization followed by primer exchange amplification (m6A-PHPEA), for the site-specific imaging of m6A methylation within cells. This approach enables high-resolution, single-cell imaging of m6A methylation across various RNA molecules coupled with efficient signal amplification. We successfully imaged three distinct m6A methylation sites concurrently in multiple cell types, revealing cell-to-cell variability in expression levels. This method promises to illuminate the dynamics of m6A-modified RNAs, potentially revolutionizing epitranscriptomic research and the development of advanced pathological diagnosis for chemical modifications.

摘要

N6-甲基腺苷(m6A)的转录后修饰对于 RNA 代谢和细胞功能至关重要。能够在单细胞水平上可视化特定位点的 m6A 甲基化,将显著增强我们对其在表观转录组学领域关键调控功能的理解。尽管如此,目前用于特定位点 m6A 的原位成像技术受到限制,这对表观转录组学研究和病理诊断构成了重大障碍。利用 DNA 杂交的精确靶向能力和 m6A 抗体的高度特异性,我们提出了一种方法,称为邻近杂交后引物交换扩增(m6A-PHPEA),用于细胞内 m6A 甲基化的特异性成像。该方法能够对各种 RNA 分子进行高分辨率、单细胞成像,并进行有效的信号放大。我们成功地在多种细胞类型中同时对三个不同的 m6A 甲基化位点进行了成像,揭示了细胞间表达水平的可变性。该方法有望揭示 m6A 修饰 RNA 的动态变化,可能会彻底改变表观转录组学研究和化学修饰的先进病理诊断的发展。

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