Multichrome encoding-based multiplexed, spatially resolved imaging reveals single-cell RNA epigenetic modifications heterogeneity

Dongsheng Mao, Xiaochen Tang, Runchi Zhang, Song Hu, Hongquan Gou, Penghui Zhang, Wenxing Li (), Qiuhui Pan (), Bing Shen () and Xiaoli Zhu ()
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Dongsheng Mao: Shanghai Tenth People’s Hospital of Tongji University
Xiaochen Tang: Shanghai Jiao Tong University School of Medicine
Runchi Zhang: Shanghai Tenth People’s Hospital of Tongji University
Song Hu: Shanghai Pudong New Area People’s Hospital
Hongquan Gou: Shanghai Tenth People’s Hospital of Tongji University
Penghui Zhang: Shanghai Pudong New Area People’s Hospital
Wenxing Li: Shanghai Tenth People’s Hospital of Tongji University
Qiuhui Pan: Shanghai Jiao Tong University School of Medicine
Bing Shen: Shanghai Tenth People’s Hospital of Tongji University
Xiaoli Zhu: Shanghai Tenth People’s Hospital of Tongji University

Nature Communications, 2025, vol. 16, issue 1, 1-16

Abstract: Abstract Understanding the heterogeneity of epigenetic modifications within single cells is pivotal for unraveling the nature of the complexity of gene expression and cellular function. In this study, we have developed a strategy based on multichrome encoding and “AND” Boolean logic recognition for multiplexed, spatially resolved imaging of single-cell RNA epigenetic modifications, termed as PRoximity Exchange-assisted Encoding of Multichrome (PREEM). Through the implementation of this strategy, we can now map the expression and nuclear distribution of multiple site-specific RNA N6-methyladenosine (m6A) modifications at the single-molecule resolution level in single-cells, and reveal the previously unknown heterogeneity. Notably, we demonstrate how these patterns change after treatment with various drugs. Moreover, cyclic imaging with tailed DNA self-assembly further suggest the scalability and adaptability of PREEM’s design. As an innovative epigenetic modification imaging tool, PREEM not only broadens the horizons of single-cell epigenetics research, enabling joint analysis of multiple targets beyond the limitations of imaging channels, but also reveals cell-to-cell variability, thereby enhancing our capacity to explore cellular functions.

Date: 2025
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DOI: 10.1038/s41467-025-56331-w

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