Abstract

The interplay between N6-methyladenosine (m6A) and oxidative stress hinges critically on m6A'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 m6A 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 m6A-modified RNA within individual cells. This approach demonstrated that oxidative stress induces substantial m6A aggregation. m6A RNA aggregation spots (ASs) showed a 65% colocalization rate with stress granules (SGs). Intriguingly, the size of m6A ASs exhibits a positive correlation with oxidative stress intensity, whereas m6A abundance demonstrates an inverse relationship. This multimodal strategy provides a comprehensive analytical framework, yielding critical insights into the regulatory dynamics of m6A modifications under oxidative stress.