Nature provides a rich toolbox of dynamic nucleic acid structures that are widespread in cells and affect multiple biological processes. Recently, non-canonical structures gained renewed scientific and biotechnological interest. One particularly intriguing form of such structures are triplexes in which a single-stranded nucleic acid molecule interacts via Hoogsteen bonds with a DNA/RNA double helix. Despite extensive research in vitro, the underlying rules for triplex formation remain debated and evidence for triplexes in vivo is circumstantial. Here, we demonstrate the development of a deep-sequencing platform termed Triplex-Seq to systematically refine the DNA triplex code and identify high affinity triplex forming oligo (TFO) variants. We identified a preference for short G-rich motifs using an oligo-library with a mix of all four bases. These high-information content motifs formed specific high-affinity triplexes in a pH-independent manner and stability was increased with G-rich double-stranded molecules. We then conjugated one high-affinity and one low-affinity variant to a VP48 peptide and studied these synthetic biomolecules in mammalian

Relevant publications:

An Oligo-Library-Based Approach for Mapping DNA-DNA Triplex Interactions In Vitro (PDF)
Beate Kaufmann, Or Willinger, Nanami Kikuchi, Noa Navon, Lisa Kermas, Sarah Goldberg and Roee Amit, 2021. ACS Synthetic Biology

Identifying triplex binding rules in vitro leads to creation of a new synthetic regulatory tool in vivo
Beate Kaufmann, Or Willinger, Noa Eden, Lisa Kermes, Leon Anavy, Oz Solomon, Orna Atar, Zohar Yakhini, Sarah Goldberg and Roee Amit. BioRxiv (2019)