Deciphering and Predicting Thermal and pH Stabilities of Triplex DNA under Multifactorial Conditions

Triplex DNA, a critical non‐canonical nucleic acid structure, plays essential roles in gene regulation, disease pathogenesis and therapeutic targeting. To unravel how environmental and sequence factors modulate triplex stability, we developed a high‐throughput five‐dimensional fluorescence resonance energy transfer melting‐annealing (5DFRETMA) method. No less than 414 sets of buffer conditions were tested. Monotonically negative correlations were observed between thermal stability and pH, as well as between pH stability and temperature. These relationships are sequence‐dependent: triplexes with higher G•C+ Hoogsteen base pair content display a higher sensitivity to pH and temperature variations. Ionic strength exerts dual effects: stabilizing the Hoogsteen hydrogen bonds in A•T base pairs while destabilizing them in G•C+ base pairs, resulting in a complex ionic strength‐dependent stability diagram. Critically, as the G•C+ content increases, the relationship between triplex stability and ionic strength shifts progressively from a monotonically positive to negative dependence. We developed predictive models that not only predict thermal stability but also pioneer the prediction of pH stability under variable pHs/temperatures and ionic environments. This work advances the mechanistic understanding of triplex DNA behavior in biologically complex settings, offering tools for the rational design of gene‐targeted therapeutics and synthetic biology applications.