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, ACS Synthethic Biology, 10, 8, 1808–1820 (2021).