Nucleic acid triplex stabilization through multimodal association

Nucleic acid triplexes are crucial for gene regulation and biotechnology, yet their formation kinetics remain poorly understood. Here, we report that dangling ends from both the triplex-forming oligonucleotide (TFO) and the duplex dramatically enhance triplex stability by accelerating association rate constants. Creating a single-base-pair dangling end at either duplex terminus or generating a TFO dangling end by removing a terminal base pair or extending a base in TFO improves binding affinity and association kinetics over blunt ends. Internal locked nucleic acid modifications also enhance stability through faster association and slower dissociation. Notably, appending a Cy3 fluorophore as a dangling end in TFO outperforms natural bases, driving both faster association and slower dissociation. Our findings establish that optimal triplex design requires strategic terminal and internal optimization, supporting a multimodal association mechanism.