Identifying triplex binding rulesin vitroleads to creation of a new synthetic regulatory toolin vivo

Abstract

Nature provides a rich toolbox of dynamic nucleic acid structures that are widespread in cells and affect multiple biological processes 1 . Recently, non-canonical structures gained renewed scientific and biotechnological interest 2,3 . One particularly intriguing form of such structures are triplexes 4 in which a single-stranded nucleic acid molecule interacts via Hoogsteen bonds with a DNA/RNA double helix 5 . Despite extensive research in vitro 6-9 , the underlying rules for triplex formation remain debated and evidence for triplexes in vivo is circumstantial 10-12 . 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 cells. Using these peptide-oligo constructs (POCs), we demonstrated possible triplex-induced down-regulation activity in 544 differentially expressed genes. Our results show that deep-sequencing platforms can substantially expand our understanding of triplex binding rules, which in turn has led to the development of a functional non-genetically encoded regulatory tool for in vivo applications.

Triplex and other DNA motifs show motif-specific associations with mitochondrial DNA deletions and species lifespan

“…What is more, this exclusion may be overly conservative. A high G-content is known to both promote G-quadruplex formation and to stabilize triplex DNA ( Buske et al 2011 , Kaufmann et al 2019 ). Although G-quadruplexes may compete with the formation of triplex DNA, this is not always so ( Sole et al 2017, del Mundo et al 2017 ), especially in mtDNA where overlapping triplex and G-quadruplex motifs can be located on opposite strands.…”

Triplex and other DNA motifs show motif-specific associations with mitochondrial DNA deletions and species lifespan

“…In contrast, both triplex and G-quadruplex motifs are strongly implicated in DNA instability. A high Gcontent is known to promote G-quadruplex formation and to stabilize triplex DNA (Buske et al 2011, Kaufmann et al 2019) thus explaining their colocalization. Although G-quadruplexes may compete with the formation of triplex DNA, this is not always so (Solé et al 2017, del Mundo et al 2017).…”

“…While multiple mature G-quadruplex detection tools exist that tend to agree with each other (Fig. S13; Lombardi and Londoño-Vallejo 2020), the choices for triplex motifs are more limited and there is little in vivo validation in mammalian genomes (Kaufmann et al 2019).…”