Probing the Intramolecular Folding of Nucleic Acids with Native Ion Mobility Mass Spectrometry: Strategies and...

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