Single-Cell Multimodal Profiling of m 6 A mRNA Modification under Oxidative Stress

The interplay between N6-methyladenosine (m 6 A) and oxidative stress hinges critically on m 6 A’s spatial localization and quantitative dynamics. Current methods are often confined to population-level analyses and struggle to incorporate multidimensional information, such as heterogeneity, spatial localization, and quantitative data. Here, we propose a multisignal integrated nanocluster-driven strategy, allowing identify m 6 A modification at location-, quantification- and single-cell-resolution. Leveraging synchrotron radiation soft X-ray microscopy, we achieve, for the first time, three-dimensional nanoscale imaging of m 6 A-modified RNA within individual cells. This approach demonstrated that oxidative stress induces substantial m 6 A aggregation. m 6 A RNA aggregation spots (ASs) showed a 65% colocalization rate with stress granules (SGs). Intriguingly, the size of m 6 A ASs exhibits a positive correlation with oxidative stress intensity, whereas m 6 A abundance demonstrates an inverse relationship. This multimodal strategy provides a comprehensive analytical framework, yielding critical insights into the regulatory dynamics of m 6 A modifications under oxidative stress.