Deciphering the RNA:DNA triplex binding code of ncRNAs and studying its interaction with chromatin

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The formation of specific triple helices, whereby an RNA strand binds to the major groove of DNA, is one means of sequence specific targeting of long and short non-coding RNA to genomic DNA. Triple helix formation is suggested to play an essential role in many nuclear processes, like antisense-mediated gene regulation, changing epigenetic states of genomic domains, regulating chromatin density, genome accessibility and recently it was suggested that the organization of the topologically associated domains and nuclear architecture is determined by a triplex-RNA mechanism. Despite the many implicated functions only little is known about the biophysical parameters of triple helix formation and its functional interaction with chromatin. Three different triple helix binding motifs exist and still no comprehensive data on sequence motifs/lengths, binding affinities, mutational impacts etc. exist, to predict binding affinities of potential motifs. We showed that motifs have very different and unexpected binding properties that currently do not allow to predict, if a poly-purine motif serves as a potential binding site in vivo. We show that many published triplex binding site predictions are wrong. Furthermore, nucleosome positioning relative to the triplex targeting site strongly affects triplex stability and we suggest that the histone post-translational modifications will further affect binding. We propose elucidating the biophysical properties of the triplex binding code and determine its interaction with chromatin to build a bioinformatic tool predicting the binding affinity of potential triplex forming elements in RNA. We think that our work presents an essential repository for addressing the mechanistical and functional impact of RNA binding to chromatin in vivo.