Isolation and genome-wide characterization of cellular DNA:RNA triplex structures I

RNA can directly bind to purine-rich DNA via Hoogsteen base pairing, forming a DNA:RNA triple helical structure that anchors the RNA to specific sequences and allows guiding of transcription regulators to distinct genomic loci. To unravel the prevalence of DNA:RNA triplexes in living cells, we have established a fast and cost-effective method that allows genome-wide mapping of DNA:RNA triplex interactions. In contrast to previous approaches applied for the identification of chromatin-associated RNAs, this method uses protein-free nucleic acids isolated from chromatin. High-throughput sequencing and computational analysis of DNA-associated RNA revealed a large set of RNAs which originate from non-coding and coding loci, including repeat elements. Combined analysis of DNA-associated RNA and RNA-associated DNA identified genomic DNA:RNA triplex structures. The results suggest that triplex formation is a general mechanism of RNA-mediated target-site recognition, which has major impact on biological functions. We captured NEAT1-associated DNA from deproteinized chromatin using a biotinylated antisense DNA oligonucleotide (ASO) that binds to NEAT1. A sense oligo that does not hybridize to NEAT1 served as control. Biological triplicates from HeLa S3 cells (rep1-3_antisense and rep1-3_sense) as well as one set from MCF7 cells were sequenced.