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.

中文翻译：

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丙烯腈介导的新生 RNA 测序，用于细胞 RNA 动力学的全转录组分析。

RNA测序极大地促进了基因表达研究，但在研究时间RNA动态方面却很薄弱；这个问题可以通过分析新生 RNA 来解决。新生 RNA 分析的一种著名方法是用非经典核苷进行代谢标记，然后进行亲和纯化，然而，纯化过程总是会给数据分析带来偏差。在这里，提出了一种用于新生 RNA 测序的化学方法，该方法避免了基于丙烯腈介导的尿苷至胞苷（U-to-C）转换（AMUC-seq）通过 4-硫尿苷（s4U）氰乙基化的亲和纯化。该方法通过 s4U 与丙烯腈的亲核加成来转换 s4U 与鸟嘌呤的碱基配对。高反应效率使得 AMUC-seq 可以直接有效地从总 RNA 中恢复新生 RNA 信息。AMUC-seq 通过用于检测 mRNA 半衰期并研究 G-四联体稳定剂（可被视为潜在的抗癌药物）在人类细胞中的直接基因靶点而得到验证。该药物的数千个直接基因靶点得到验证（这些基因在SRC和HRAS等癌症中显着富集）。AMUC-seq 还证实了 G-四链体稳定性会影响 RNA 多腺苷酸化。这些结果表明 AMUC-seq 适合研究时间 RNA 动力学，并且它可以成为研究转录调节药物治疗机制的一种有前途的策略。