Transcriptome-wide mapping reveals an RNA-dependent mechanism of platinum cancer drug [Plat-RNAseq cPDS]

Clinically used small molecules has been predicted to off-target to RNAs. However, the extend of this interaction is small molecule cancer therapeutics has not been characterized. Here, we screened for anticancer smalll molecules for their RNA interactions and uncovered widespread RNA off-targeting. Cisplatin was found to be one among the RNA binders. We used it as a model agent to identify the specific transcripts it interact with. We developed Plat-RNAseq, a click-chemistry-mediated transcriptome-wide assay to map transcriptome-wide interaction of cisplatin. Our results revealed that cisplatin binding was enriched at guanine-rich regions capable of forming RNA G-quadruplexes (rG4s). We use an rG4 ligand, carboxy-Pyridostatin (cPDS), to block access of platin-drug to rG4. This experiment test whether cPDS blocked platinum drug accumulation on cellular RNAs. Overall design: Cells were treated with cPDS (5 µM) or DMF for 6 hours followed by 1,3-platin (30 µM). The cells were harvested 18 hours after 1,3-platin treatment and proecessed according to the Plat-RNAseq protocol described below. The goal of the experiment is to test whether rG4 ligands can influence the RNA binding of platinum drugs.

临床应用的小分子化合物已被预测可与核糖核酸(RNA)发生脱靶相互作用。然而，这类相互作用在小分子抗癌疗法中的覆盖程度尚未被阐明。本研究针对抗癌小分子化合物与RNA的相互作用开展筛选，发现了广泛存在的RNA脱靶结合现象。顺铂(Cisplatin)被鉴定为这类RNA结合分子之一，我们以其作为模型分子，鉴定其结合的特异性转录本。我们开发了Plat-RNAseq技术——一种基于点击化学(click chemistry)的全转录组检测方法，用于绘制顺铂在全转录组范围内的结合相互作用图谱。研究结果显示，顺铂的结合位点富集于可形成RNA G-四链体(RNA G-quadruplexes, rG4s)的富鸟嘌呤区域。我们使用一种RNA G-四链体配体——羧基吡啶司他汀(carboxy-Pyridostatin, cPDS)，来阻断铂类药物与RNA G-四链体的结合，本实验旨在验证cPDS是否可阻断铂类药物在细胞RNA上的富集。实验设计：将细胞用浓度为5μM的cPDS或二甲基甲酰胺(Dimethylformamide, DMF)处理6小时，随后再用30μM的1,3-铂(1,3-platin)处理；在1,3-铂处理18小时后收集细胞，并按照下文所述的Plat-RNAseq实验流程进行样本处理。本实验的核心目标为验证RNA G-四链体配体是否可影响铂类药物与RNA的结合。