Triplex-forming oligonucleotides (TFOs) enable sequence-specific recognition of polypurine-containing duplex DNA through non-covalent interactions and hold considerable promise for antigene and biotechnology applications. However, triplex stability is highly sequence-dependent and is often disrupted by pyrimidine interruptions and competing duplex equilibria, limiting broader use. Robust analytical approaches are therefore required to characterise and stabilise triplex assemblies under near-physiological conditions. Building on our work in structural mass spectrometry (MS), we apply native MS-centred workflows to interrogate DNA–DNA non-covalent complexes and guide rational chemical modification strategies for enhanced triplex formation.
Native electrospray ionisation mass spectrometry and ion mobility–mass spectrometry were used to resolve stoichiometry, population distributions, thermal stability, and conformational features of triplex assemblies formed between duplex targets and modified TFOs. Complementary UV–Vis spectroscopy and isothermal titration calorimetry quantified melting transitions, binding affinities, and thermodynamics. Chemically modified oligonucleotides incorporating locked nucleic acid (LNA), backbone spacers, and base substitutions were synthesised via oxidative amination and thiol-alkylation to evaluate their effects on stability across model and biologically relevant bacterial sequences. Native MS directly resolved co-existing duplex and triplex populations and revealed sequence- and modification-dependent differences in assembly not observable by traditional ensemble methods. LNA substitutions markedly increased triplex abundance and enabled formation in interruption-containing targets where unmodified TFOs failed. Isothermal titration calorimetry provided label-free quantification of triplex thermodynamics and kinetics, confirming slower and weaker Hoogsteen-mediated binding relative to duplex formation. Ion mobility measurements delivered collision cross-section data for conformational assignment and enabled separation of overlapping species.
Together, these results demonstrate that structural MS wiht complementary biophysical techniques can directly resolve equilibrating nucleic acid assemblies and guide MS-informed chemical modification strategies to stabilise DNA triplexes in biologically relevant sequence contexts. This integrated analytical framework establishes structural MS as a powerful platform for the rational design of stable DNA triplex assemblies and future antigene and biomolecular complex applications.