Native Mass Spectrometry and Nucleic Acid G-Quadruplex Biophysics: Advancing Hand in Hand

Abstract

While studying nucleic acids to reveal the weak interactions responsible for their three-dimensional structure and for their interactions with drugs, we also contributed to the field of biomolecular mass spectrometry, both in terms of fundamental understanding and with new methodological developments. A first goal was to develop mass spectrometry approaches to detect non-covalent interactions between antitumor drugs and their DNA target. Twenty years ago, our attention turned towards specific DNA structures such as the G-quadruplex (a structure formed by guanine-rich strands). Mass spectrometry allows to discern which molecules interact with one another by measuring the masses of the complexes, and quantity the affinities by measuring their abundance. The most important findings came from unexpected masses. For example, we showed the formation of higher- or lower-order structures by G-quadruplexes used in traditional biophysical assays. We can also derive complete thermodynamic and kinetic description of G-quadruplex folding pathways by measuring cation binding, one at a time. Getting quantitative information required accounting for nonspecific adduct formation and for the response factors of the different molecular forms. But with these caveats in mind, the approach is mature enough for routine biophysical characterization of nucleic acids. A second goal is to obtain more detailed structural information on each of the complexes separated by the mass spectrometer. One such approaches is ion mobility spectrometry, and even today the challenge lies in the structural interpretation of the measurements. We showed that, although structures such as G-quadruplexes are well-preserved in the MS conditions, double helices actually get more compact in the gas phase. These major rearrangements forced us to challenge comfortable assumptions. Further work is still needed to generalize how to deduce structures in solution from ion mobility spectrometry data, and in particular how to account for the electrospray charging mechanisms and for ion internal energy effects. These studies also called for complementary approaches to ion mobility. Recently, we applied isotope exchange labeling mass spectrometry to characterize nucleic acid structures for the first time, and we reported the first ever circular dichroism ion spectroscopy measurement on mass-selected trapped ions. Circular dichroism plays a key role in assigning the stacking topology, and our new method now opens the door to characterizing a wide variety of chiral molecule by mass spectrometry. In summary, advanced mass spectrometry approaches to characterize gas-phase structures work well for G-quadruplexes because they are stiffened by inner cations. The next objective will be to generalize these methodologies to a wider range of nucleic acid structures.