biostudies-literatureUnknown5(7)Wang LWestlake UniversityRNA structure plays a crucial role in its function and undergoes dynamic changes throughout its life cycle. To study these dynamics, we developed TAS sequencing (TAS-seq), which expresses the deaminase TadA-8e in specific subcellular compartments to modify single-stranded adenosines, particularly within hairpin loops. We applied TAS-seq to the nucleus, cytosol, and endoplasmic reticulum membrane, identifying adenosine structural variations and compartment-specific regulation of RNA stability. Single-cell TAS-seq revealed structural heterogeneity of cytosolic RNAs. Additionally, adenosines labeled by TAS-seq contribute to guide RNA optimization in the CRISPR-Cas13d system. Our method provides insights into compartment-specific RNA structural dynamics, cell-specific heterogeneity, and their Cell reports methods101087https://www.ebi.ac.uk/biostudies/studies/S-EPMC12296453biostudies-literatureTAS-seq enables subcellular single-stranded adenosine profiling by signal peptide-assisted adenosine deamination.PMC12296453Ma LWu PLu ZZhou YYu ZWang LfalseTAS-seq enables subcellular single-stranded adenosine profiling by signal peptide-assisted adenosine deamination.RNA structure plays a crucial role in its function and undergoes dynamic changes throughout its life cycle. To study these dynamics, we developed TAS sequencing (TAS-seq), which expresses the deaminase TadA-8e in specific subcellular compartments to modify single-stranded adenosines, particularly within hairpin loops. We applied TAS-seq to the nucleus, cytosol, and endoplasmic reticulum membrane, identifying adenosine structural variations and compartment-specific regulation of RNA stability. Single-cell TAS-seq revealed structural heterogeneity of cytosolic RNAs. Additionally, adenosines labeled by TAS-seq contribute to guide RNA optimization in the CRISPR-Cas13d system. Our method provides insights into compartment-specific RNA structural dynamics, cell-specific heterogeneity, and their 2025-01-01T00:00:00Z2025 Jul2026-07-15T05:59:56.341Z2025-08-13T03:04:14.159ZS-EPMC122964534057083810.1016/j.crmeth.2025.101087