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.
Highlights
Introduction weak in studying temporalRNA dynamics; this issue can be addressedRNA sequencing (RNA-seq) is a revoluby analyzing nascent RNAs
We presented a new chemical method for enrichment-free nascent RNA sequencing
Our experiments demonstrated that acrylonitrile-mediated s4U-to-C mutation permits the recognition of metabolically labeled RNA at 1 bp resolution without additional RNA-purification processes
Summary
We predicted that the original N3 of s4U is no longer a competent proton donor after cyanoethylation, leading to s4U inhibiting Watson–Crick base pairing with A. Instead, it forms non-canonical base pair with G. By adjusting the reaction conditions, the reaction could be completed within hours (Figure S2A,B, Supporting Information). We compared the selectivity and reactivity of our method with the reported ones (SLAM-seq[10] and TimeLapse-seq[11]) all these three methods can achieve high reaction selectivity and efficiency (Figure S3, Supporting Information). Only dGTP incorporation was observed opposite to the ces4URNA template even after a long reaction time (Figure S2C, Supporting Information). S4U cyanoethylation resulted in a thoroughly s4U-to-C conversion in the base-pairing manner
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