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Negative Electrospray Supercharging Mechanisms of Oligonucleotide Single Strands and G-quadruplexes

When sprayed from physiological ionic strength, nucleic acids typically end up with low levels of charging and in compact conformations. Increasing the electrospray negative charging of nucleic acids while preserving the native non-covalent interactions can help distinguish solution folds by ion mobility mass spectrometry. To get fundamental insight into the supercharging mechanisms of nucleic acids in the negative mode, we studied model G-quadruplex structures and single strand controls in 100 mM ammonium acetate. We found that adding 0.4% propylene carbonate, 0.4% sulfolane or 0.1% m-NBA induces native supercharging through the charged residue mechanism. However, although 0.4% m-NBA shows the highest supercharging ability, it induces unwanted unfolding of solution-folded G-quadruplexes. The supercharging effect resembles the effect of lowering the ionic strength, which could be explained by partial neutralization of the ampholytes when droplets become more concentrated in their non-aqueous components. The supercharging ability ranks: PC < sulfolane < m-NBA. m-NBA adducts to G-quadruplexes with high charge states confirms that the supercharging agent interacts directly with DNA. Surprisingly, in presence of supercharging agents, more negative charge states also bear more alkali metal ion adducts. This suggests that native supercharging results from larger droplets evaporating to the charged residue, leading to higher concentration of both the supercharging agent and of alkali counterions. However, when negative charge carriers from the electrolyte become too rare, chain ejection accompanied by denaturation, and hence non-native supercharging, can become predominant.