An Oligo-Library-Based Approach for Mapping DNA-DNA Triplex Interactions In Vitro.
We present Triplex-seq, a deep-sequencing method that systematically maps the interaction space between an oligo library of ssDNA triplex-forming oligos (TFOs) and a particular dsDNA triplex target site (TTS). We demonstrate the method using a randomized oligo library comprising 67 million variants, with five TTSs that differ in guanine (G) content, at two different buffer conditions, denoted pH 5 and pH 7. Our results show that G-rich triplexes form at both pH 5 and pH 7, with the pH 5 set being more stable, indicating that there is a subset of TFOs that form triplexes only at pH 5. In addition, using information analysis, we identify triplex-forming motifs (TFMs), which correspond to minimal functional TFO sequences. We demonstrate, in single-variant verification experiments, that TFOs with these TFMs indeed form a triplex with G-rich TTSs, and that a single mutation in the TFM motif can alleviate binding. Our results show that deep-sequencing platforms can substantially expand our understanding of triplex binding rules and aid in refining the DNA triplex code.
['Kaufmann|Beate|B|', 'Willinger|Or|O|', 'Kikuchi|Nanami|N|', 'Navon|Noa|N|', 'Kermas|Lisa|L|', 'Goldberg|Sarah|S|', 'Amit|Roee|R|0000-0003-0580-7076']
triplex oligo library TFO TTS EMSA Shannon entropy anti-parallel triplex next-generation sequencing parallel triplex