Identification of Triple-helix DNA Motifs by CuAAC Click Reaction

Triplexes are noncanonical DNA structures, in which a single-stranded DNA or RNA binds to a double-stranded DNA molecule via Hoogsteen base-pairing. In recent studies, bioinformatic analyses have revealed that regulatory elements such as promoters and enhancers are enriched with potential triplex targeting sites, suggesting potential triplex formation in transcriptional regulation with non-coding RNA. Yet, the mechanism of DNA:RNA triplex formation, how triplex formation is regulated, and how triplexes impact gene expression remain unknown.

To address these questions and better understand triplexes, we first looked into the underlying algorithm of triplex motif. A pool of triplex-forming oligonucleotide target sequences (TTS) and triplex-forming oligonucleotides (TFO) were incubated in vitro. Upon formation of triplex structure, TTSs and TFOs were covalently linked by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and analyzed using high throughput sequencing. In the same way, 5-ethynyl-2-deoxyuridine (EdU) was incorporated in U2OS and clicked upon triplex formation. We seek to validate triplex formation in vivo by transfecting the best candidate TFO obtained from computational analysis into the cell nucleus.

Aside from the biological implications, potential applications in developing triplex based molecular biology tools and therapies are attracting broad interest among the scientific communities. In the future, we plan on using the triplex motif not only to answer the fundamental questions regarding the triplex, but to engineer single-stranded oligonucleotide that can target specific dsDNA, which could be the base for dsDNA detection, drug delivery, imaging, and gene regulation.