| Summary: | Abstract RNA sequencing has greatly facilitated gene expression studies but is weak in studying temporal RNA dynamics; this issue can be addressed by analyzing nascent RNAs. A famous method for nascent RNA analysis is metabolic labeling with noncanonic nucleoside followed by affinity purification, however, purification processes can always introduce biases into data analysis. Here, a chemical method for nascent RNA sequencing that avoids affinity purification based on acrylonitrile‐mediated uridine‐to‐cytidine (U‐to‐C) conversion (AMUC‐seq) via 4‐thiouridine (s4U) cyanoethylation is presented. This method converts s4U base‐pairing with guanine through the nucleophilic addition of s4U to acrylonitrile. The high reaction efficiency permits AMUC‐seq directly and efficiently to recover nascent RNA information from total RNAs. AMUC‐seq is validated by being used to detect mRNA half‐lives and investigating the direct gene targets of a G‐quadruplex stabilizer, which can be regarded as potential anticancer drug, in human cells. Thousands of direct gene targets of this drug are verified (these genes are significantly enriched in cancer such as SRC and HRAS). AMUC‐seq also confirms G‐quadruplex stabilization that impacts RNA polyadenylation. These results show AMUC‐seq is qualified for the study of temporal RNA dynamics, and it can be a promising strategy to study the therapeutic mechanism of transcription‐modulating drugs.
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