Rodrigo Maldonado et al., (2017) RNA; PMID: 29222118

Triplexes are noncanonical DNA structures, which are functionally associated with regulation of gene expression through ncRNAtargeting to chromatin. Based on the rules of Hoogsteen base-pairing, polypurine sequences of a duplex can potentially formtriplex structures with single-stranded oligonucleotides. Prediction of triplex-forming sequences by bioinformatics analyseshave revealed enrichment of potential triplex targeting sites (TTS) at regulatory elements, mainly in promoters and enhancers,suggesting a potential function of RNA–DNA triplexes in transcriptional regulation. Here, we have quantitatively evaluated thepotential of different sequences of human and mouse ribosomal RNA genes (rDNA) to form triplexes at different salt and pHconditions. We show by biochemical and biophysical approaches that some of these predicted sequences form triplexes withhigh affinity, following the canonical rules for triplex formation. We further show that RNA triplex-forming oligos (TFOs) aremore stable than their DNA counterpart, and point mutations strongly affect triplex formation. We further show differentialsequence requirements of pyrimidine and purine TFO sequences for efficient binding, depending on the G–C content of theTTS. The unexpected sequence specificity, revealing distinct sequence requirements for purine and pyrimidine TFOs, showsthat in addition to the Hoogsteen pairing rules, a sequence code and mutations have to be taken into account to predictgenomic TTS.