PROJECT SUMMARY The goal of this research proposal is to develop new technologies for the identification of regions within long non-coding RNAs that engage in R-loop and RNA-triplex formation. Long non-coding RNAs (lncRNAs) can localize to chromatin and regulate important nuclear processes such as transcription, recombination, and repair. LncRNAs are targeted to chromatin through their association with specific proteins or they can interact directly with the DNA in chromatin. The extent to which lncRNAs directly contact DNA and the RNA regions responsible for these connections are largely unknown. We will bridge this gap in knowledge by developing new genomics technologies that will yield high-resolution maps of regions within all nuclear RNAs that interact with DNA through R-loop and triplex structures. RNA-DNA interactions on chromatin occur through the formation of two distinct structures: 1) R-loops that form through Watson-Crick base pairing between RNA and its complementary DNA strand, and 2) RNA-triplexes that form because an RNA occupies the major groove of the double helix and forms Hoogsteen or reverse Hoogsteen hydrogen bonds with the purines in the Watson-Crick DNA strands. Antibody- dependent and -independent strategies exist to identify the genome-wide distribution of R-loop structures. However, most of these methods sequence the DNA component of R-loops. While information from the perspective of DNA provides a view of where R-loops form across the genome, it cannot answer whether R- loops at specific genomic regions form as a by-product of transcription or because of the presence of regulatory non-coding RNAs. Moreover, the existing antibody-dependent approach to recover the RNA component of R- loops lacks sensitivity, specificity, and resolution. Knowledge of the RNA component of R-loops will allow us to distinguish regulatory R-loops, which may involve lncRNAs, from co-transcriptional R-loops and discover new R-loop based mechanisms in genome regulation. The need for new tools to identify RNA-triplexes is more urgent. While computational approaches to predict triplex formation have been developed, genomic methods to detect endogenous triplexes in vivo do not exist. New methods to detect RNAs within R-loops and RNA-triplexes will enhance our understanding of these structures and identify the extent of their function in gene regulation. These tools can be applied to examine how coding and non-coding RNAs differ in their interactions with chromatin and can help define new principles for gene regulation through RNA interactions. Here, we propose to develop new antibody-independent strategies that employ epitranscriptomics to enrich for and identify the RNA component of R-loops and RNA-triplexes.