Novel Insight into Proximal DNA Domain Interactions from Temperature-Controlled Electrospray Ionization Mass Spectrometry

Quadruplexes are non-canonical nucleic acid structures essential for many cellular processes. Hybrid quadruplex-duplex oligonucleotide assemblies comprised of multiple domains are challenging to study with conventional biophysical methods due to their structural complexity. Here, we introduce a novel method based on native mass spectrometry (MS) coupled with a custom-built temperature-controlled nanoelectrospray ionization (TCnESI) source designed to investigate interactions between proximal DNA domains. Thermal denaturation experiments were aimed to study unfolding of multi-stranded oligonucleotide constructs derived from biologically relevant structures and to identify unfolding intermediates. Using the TCnESI MS, we observed changes in T_m and thermodynamic characteristics of proximal DNA domains depending on the number of domains, their position, and order in a single experiment.