Sequence-specific recognition of double-stranded DNA (dsDNA) can be achieved using a variety of natural and artificial oligonucleotides that bind to dsDNA via non-covalent interactions. Among these, triplex-forming oligonucleotides (TFOs) enable the formation of triple-stranded DNA structures (triplexes), which hold promise for applications in molecular biology, biotechnology, and therapeutics. However, the sequence dependence of triplex stability limits their applicability. To address this, chemically modified TFOs have been developed to enhance triplex stability, with their design and evaluation relying on synthetic, analytical, and computational approaches. Despite these advancements, the lack of a standardised methodology for the evaluation of triplex formation hinders the rational design of novel TFOs.
Building on previous research from our group, this study examines the advantages of native mass spectrometry (nMS) in combination with traditional analytical methods such as UV-vis spectroscopy, non-denaturing gel electrophoresis, and isothermal titration calorimetry. By assessing the strengths and limitations of each technique, we demonstrate that a combination of complementary methods is necessary for a comprehensive characterisation and comparison of triplexes. Notably, we show that nMS not only provides mass-resolved insights into solution-phase species but also reveals the presence of species undetectable by other methods. This additional level of detail improves our understanding of DNA-DNA interactions and sequence-specific effects, offering deeper insight into the complex equilibria governing these systems. Furthermore, the use of ion mobility-mass spectrometry (IM-MS) provides valuable collision cross-section data, enhancing our understanding of structural conformations in the gas phase, and enables further separation of charge states of different species with overlapping m/z values.
With these findings, we aim to provide a comprehensive perspective on DNA triplex formation and establish a robust, standardised analytical methodology. Progress towards this goal will be discussed, along with a critical evaluation of issues pertaining to current methodologies.