Ion mobility spectrometry reveals intermediate states in temperature-resolved DNA unfolding

•Stable intermediate DNA unfolding states revealed.•Solution phase unfolding of double stranded DNA followed with ion mobility MS.•Temperature resolved ion mobility with new temperature controlled ESI Source.

Electrospray ionization (ESI) mass spectrometry (MS) is used to study the non-covalent interactions of short 12-mer oligonucleotides under native conditions. A home-built, temperature-controlled ESI source is employed to elevate the spray temperature above the melting temperature (Tm) of the DNA duplexes, enabling the study of DNA interactions in the liquid phase rather than gas phase. Ion mobility spectrometry (IMS)-MS is used to investigate the 3-dimensional structure of the hybridized oligonucleotides and to track conformational changes in the oligonucleotide duplexes during temperature-induced melting in solution. Results show two additional drift times at T > 35 °C, indicating 2-fold (partial) unfolding dynamics for the DNA duplex with stable intermediates.

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