Triplexforming

Triplexforming refers to the formation of a three-stranded DNA structure in which a single-stranded nucleic acid binds to a pre-existing DNA duplex through Hoogsteen or reverse Hoogsteen hydrogen bonds. The binding third strand, often called a triplex-forming oligonucleotide (TFO), typically targets a duplex that contains a long run of homopurine–homopyrimidine base pairs, creating two lattice-like base-triples instead of the usual two.

There are two principal binding motifs. Pyrimidine-rich TFOs tend to bind in parallel orientation to the purine-rich strand, forming T•A–T and C+–G–C triplets, with cytosine often requiring protonation to stabilize the C+ motif. Purine-rich TFOs can form reverse Hoogsteen triplets in antiparallel arrangements, expanding the range of possible target sequences but often with different stability and specificity. Chemical modifications and alternative backbones (such as peptide nucleic acids, PNAs, or locked nucleic acids, LNAs) are used to improve affinity and broaden the usable pH range.

Target specificity is a major consideration, as triplex formation requires extended homopurine–homopyrimidine tracts and can be sensitive to DNA conformation and chromatin context. Off-target interactions and limited accessibility in vivo can reduce effectiveness. Nonetheless, triplex-forming strategies have been explored to regulate transcription, impede replication, or direct site-specific recombination or mutagenesis.

In practical use, researchers employ TFOs and their analogs to study gene regulation and as potential tools for genome engineering. Ongoing developments aim to improve stability, delivery, and sequence flexibility to extend their applicability in research and therapeutic contexts.