Transcriptome-wide mapping reveals an RNA-dependent mechanism of platinum cancer drugs [CUT&TAG]

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). To test the relationship with RNA-DNA hybrid R-loop structures and cisplatin-RNA interaction, we used CUT&Tag experiment using S9.6 R-loop specfic antibody. This result revealed an increased cisplatin interacitn on R-loop engaged RNAs. Overall design: Cells were treated with Cisplatin (25 µM) or DMF for 6 hours. R-loops were pulldown and sequencing libraries were prepared using CUT&Tag-IT R-loop Assay Kit (Active Motif, 53167). The goal was to estimate R-loop enrichment in cisplatin-target RNA promoters.