Repeated DNA sequences account for more than 50 % of our genome. These sequences can fold into non-canonical DNA structures playing essential roles in cellular function. Guanine-rich direct repeats can form G-quadruplexes (GQs) and partially complementary strands can form branch-like structures, such as three-way junctions (TWJs). Misfolding or persistence of these structures can lead to DNA genomic instability causing severe cellular damage, apoptosis, or abnormal cell growth. To develop ligands that specifically target these structures, detailed information about binding sites, affinity, thermodynamics, and structure aid in developing targeted genetic disease therapeutics. The challenges using conventional methods arise from the structural complexity and variability of these structural assemblies. This can be overcome by temperature-controlled nanoelectrospray ionization (TC-nESI), a method to simultaneously analyze individual forms of DNA or DNA-ligand complexes and characterize their thermodynamics.¹ Here we introduce a custom-built TC-nESI source, which is designed such as the nESI emitter is placed between two copper blocks containing a Peltier element, which guarantees uniformly distributed heat and allows for temperature gradients between 13 – 90 °C.

MS thermal denaturation experiments were designed to acquire mass spectra with increasing source temperature and to observe unfolding steps of individual intermediates of multi-stranded oligonucleotide constructs. We observed changes in melting temperatures (T_m) of individual DNA domains depending on the type of domain, the number of domains, their position, the presence of ligand, and more. Following a detailed van’t Hoff analysis, the changes can also be used to calculate thermodynamic parameters.

A DNA TWJ is a complex comprising three double helices, half-complementary to each other, which converge toward a central branchpoint (the potential ligand binding site).² TCnESI analysis of TWJ confirmed an unfolding pathway with three different duplex intermediates. Van’t Hoff analyses of MS thermal denaturation experiments showed a stabilizing effect of additional base pairing in overhangs of intermediates. We propose that the competition between the formation of the duplex and the TWJ (represented by ∆G°) depends on the formation of additional base pairs between overhanging strands. Effects of the TWJ-binding ligands on the unfolding pathway and thermodynamic stability were also investigated. These ligands bind specifically to the cavity in the center of the branchpoint and greatly affect the TWJ unfolding thermodynamics.

In conclusion, our TC-nESI methodology is a powerful tool for identifying binding events, characterizing their thermodynamic aspects, and quantifying unfolding intermediates in the wide temperature range. Therefore, TC-nESI is a promising method to fill up the gaps in the current therapeutic drug development and be a powerful alternative to current methods.