Probing the Intramolecular Folding of Nucleic Acids\nwith Native Ion Mobility Mass Spectrometry: Strategies and Caveats

The\ngoal of native mass spectrometry is to obtain information on\nnoncovalent interactions in solution through mass spectrometry measurements\nin the gas phase. Characterizing intramolecular folding requires using\nstructural probing techniques such as ion mobility spectrometry. However,\ninferring solution structures of nucleic acids is difficult because\nthe low-charge state ions produced from aqueous solutions at physiological\nionic strength get compacted during electrospray. Here we explored\nwhether native supercharging could produce higher charge states that\nwould better reflect solution folding, and whether the voltage required\nfor collision-induced unfolding (CIU) could reflect preserved intramolecular\nhydrogen bonds. We studied pH-responsive i-motif structures with different\nloops, and unstructured controls. We also implemented a multivariate\ncurve resolution procedure to extract physically meaningful pure components\nfrom the CIU data and reconstruct unfolding curves. We found that\nthe relative unfolding voltages reflect to some extent, but not always\nunambiguously, the number of intramolecular hydrogen bonds that were\npresent in solution. Reaching phosphate charging densities over 0.25\nmakes it easier to discriminate between structures, and the use of\nnative supercharging agents is thus essential. We also uncovered several\ncaveats in data interpretation: (1) when different structures (for\nexample the i-motif with and without hairpin) unfold via different\npathways, the unfolding voltages do not necessarily reflect the number\nof hydrogen bonds, (2) unstructured controls also undergo unfolding,\nand the base composition influences the unfolding voltage, (3) changing\nthe solution pH also unexpectedly changed the unfolding voltage, and\n(4) the ion mobility patterns become more complicated when two structures\nare present simultaneously, such as an i-motif and a harpin, because\nof opposite effects on the collision cross section upon activation.