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

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 have established methods (DNA-IP and SPRI) to enrich RNA regions that are directly associated with DNA (i.e., TriplexRNAs). These RNAs were sequenced as biological replicates (IP_rep1-3 and SPRI_rep1-4). Nuclear and chromatin-associated RNAs were sequenced as control (nuclear_rep1-2 and chromatin_rep1-4). For U2OS cells, biological duplicates were sequenced from TriplexRNA (IP_rep1-2) and nuclear RNA (nuclear_rep1-2).