This work focuses on enhancing the stability of DNA triplex structures through chemical modification and native mass spectrometry analysis. Triplex-forming oligonucleotides (TFOs) hold promise for antigene therapy, but their biological application is limited by sequence interruptions in triplex target sites and reduced structural stability. By integrating backbone and base modifications with high-resolution native mass spectrometry, we aim to observe and improve stability and optimize triplex formation under physiologically relevant conditions. These insights provide a foundation for designing robust TFOs with therapeutic potential against bacterial gene targets.
Native electrospray ionization mass spectrometry (ESI-MS) was used to monitor triplex formation and stoichiometry in model targets and biologically relevant bacterial sequences containing pyrimidine interruptions. Complementary UV-Vis spectroscopy and isothermal titration calorimetry (ITC) quantified base stacking, binding affinities, and thermodynamics in physiological buffers. Oligonucleotides were engineered via oxidative amination and thiol alkylation of 4-thioldioxyuracil to install alkyne/azide handles using click chemistry, enabling systematic backbone and base modifications with minimal steric penalty. TFOs included DNA-only, dSpacer, and locked nucleic acid (LNA) variants. Spectra were acquired on a high-resolution Q-TOF platform in native conditions, and data were interpreted to resolve triplex vs duplex populations and charge-state distributions.
We used native MS to directly monitor assembly stoichiometry in order to test chemical modification strategies that overcome biologically prevalent pyrimidine interruptions in triplex target sites. Locked nucleic acid (LNA) substitutions within triplex‑forming oligonucleotides (TFOs) markedly increased triplex abundance relative to unmodified DNA or dSpacer variants.1 This improvement was evident even in the sequences containing destabilizing cytosine interruptions where DNA‑only and dSpacer TFOs failed.1 Increasing the number of LNA bases further improved triplex yields, with UV-Vis melting experiments showing triplex melting temperatures values approaching duplex stability in the most stabilized systems. These data demonstrate that rational backbone/base modification, particularly alternating LNA/DNA designs enables robust triplex formation in biologically relevant bacterial targets, thereby expanding antigene applicability under near‑physiological buffers.1
Complemented by MS, isothermal titration calorimetry (ITC), has provided the first quantitative, label‑free determination of both thermodynamic and kinetic parameters for DNA triplex assembly in solution. ITC resolved two sequential binding events, initial duplex formation followed by triplex formation, within a single experiment, allowing extraction of Ka, ΔG, ΔH, ΔS and the association/dissociation rate constants for each step.2 Triplex formation was consistently slower and less stable than duplex formation, reflecting weaker Hoogsteen interactions and the higher electrostatic penalty upon introducing a third strand.2 We further showed that strand length modulates both stability and dynamics where longer strands produced higher affinities and lower koff, whereas shorter strands exhibited faster dissociation and reduced triplex stability. Sequence composition also mattered as guanine substitutions enhanced base stacking and measurably altered the enthalpy and kinetics of the second (triplex) event.2 Finally, acidic pH strengthened triplex formation via cytosine protonation, highlighting opportunities for pH‑responsive designs.2
Together, our ITC, MS and UV–Vis framework establishes a mechanistic map for optimising triplex assembly with using ITC to quantify how length, sequence, and pH shape energetics and kinetics. Then apply native mass spectrometry to confirm specific stoichiometries while guiding chemically modified TFO designs that overcome sequence interruptions in target sites.