Develop a technology for whole-genome sequencing of RNA-DNA triplexes

About This Grant

PROJECT SUMMARY: Yu Liu, PhD (Principal Investigator; PI) LncRNAs are >200nt RNA molecules that do not translate into protein products. The number of lncRNAs is far greater than their protein-coding counterparts, but the functions and underlying mechanisms of lncRNAs are less understood. A few lncRNAs modulate the expression of their target genes through forming an RNA-DNA triple helix at gene regulatory regions such as enhancers and promoters; this new layer of gene regulation is particularly important for developmental gene expression programs. However, knowledge about this new regulatory mechanism is scarce. The research community mainly relies on candidate lncRNA-based methods, not unbiased whole-genome approaches. The major technological challenge is that we cannot reliably capture whole-genome RNA-DNA triplexes in vivo. We propose to capture RNA-DNA triplex by using the differential affinity of Thiazole Orange (TO) to complex helical structures vs. canonical structures. TO is a classic fluorochrome for double-stranded DNA (dsDNA), but its affinity to triplex and quadruplex structures of DNA is substantially higher. Because RNA-DNA triplex is structurally comparable to, but more stable than DNA triplex, we predict that we can use TO to identify genomic RNA-DNA triplexes. The core of this new technology, TO-Triplex-Seq, comprises Biotin-conjugated-TO labelling and subsequent Avidin-pulldown, followed by DNA purification and sequencing. We will distinguish RNA-DNA triplex from other complex helical structures using special RNAse treatments in parallel chromatin samples. The unique strength of this new technology is that we will specifically capture RNA-DNA triplexes, not other complexes in which RNA and DNA form a duplex (R-loop) or indirectly interact through other binding partners. We will prove the concept and validate this new technology with two specific aims. SA1 will determine the affinity between TO and synthetic RNA-DNA triplexes and test the framework of pulling-out RNA-DNA triplexes from a pool containing RNA-DNA triplexes, DNA-only triplexes and dsDNAs. SA2 will establish and validate the new technology using a multi-scale approach. We will (1) characterize the sequence features of the RNA-DNA triplex sites; (2) study how triplex formation changes during active and suppressed rRNA biogenesis which is regulated by a pRNA-rDNA triplex; (3) study whether distinct sets of lncRNAs contribute to triplex formation in undifferentiated and differentiated hESCs; (4) further annotate the RNA-DNA triplex datasets to publicly available chromatin state datasets. At the conclusion of this project, we expect to publish a detailed protocol which others can easily adopt and further build upon. Ultimately, we plan to develop a TO-labeling- based multiomics toolset that comprehensively describes the RNA, DNA, and even protein components (if present) of RNA-DNA triplexes and hence contributing to the understanding of transacting lncRNAs.