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

Abstract

Conspectus 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 noncovalent interactions between antitumor drugs and their DNA target. Twenty years ago, our attention turned toward specific DNA structures such as the G-quadruplex (a structure formed by guanine-rich strands). Mass spectrometry allows one to discern which molecules interact with one another by measuring the masses of the complexes, and quantify 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 also derived complete thermodynamic and kinetic description of G-quadruplex folding pathways by measuring cation binding, one at a time. Getting quantitative information requires accounting for nonspecific adduct formation and for the response factors of the different molecular forms. With these caveats in mind, the approach is now 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 approach 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 spectrometry. 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 molecules 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.

A biophysical study of the interactions of palladium(ii), platinum(ii) and gold(iii) complexes of aminopyridyl-2,2′-bipyridine ligands with RNAs and other nucleic acid structures

“…In fact, the ionic radii of these two ions are very similar (1.48 and 1.33 Å, respectively) and lead to the same placement of these cations between the tetrads' planes. 55,56 The ESI mass spectrum was then recorded in order to verify the proper formation of the G4 structure. The spectrum (see Fig.…”

“…S14 †) shows a signal cluster at 6937.358, 6954.347, 6975.375 and 6998.323 Da that was in perfect agreement with the CTA-22 molecular mass plus up to four ammonium ions, which promote the G4 assembly and are accommodated inside the structure. 52,[56][57][58][59] The presence of these ammonium ions is straightforward information that the G4 structure was present and retained during the electrospray ionisation process. Beyond this cluster, a much less intense peak was still visible at 6920.322 Da, corresponding to a residual amount of the CTA-22 single-stranded DNA.…”

The combination of laser photodissociation, action spectroscopy, and mass spectrometry to identify and separate isomers

“…Adapted with permission from V. Gabelica, Accounts of Chemical Research, 2021, 54, 3691-3699. 228 This journal is © The Royal Society of Chemistry 2022 technical advances are required before isomer separation and disentanglement become routine. In this Feature Article, we focused on the techniques for distinguishing isomers that involve UV and visible photodissociation with mass spectrometry.…”

Template-Assembled Synthetic G-Quartets (TASQs): multiTASQing Molecular Tools for Investigating DNA and RNA G-Quadruplex Biology

“…Globally speaking, PNA DOTASQ was considered as a pan-G4 interacting agent as it did interact with the G quartet whatever the G4 to which it belonged. These G4-interacting properties were satisfying enough to go further than simple G4-affinity measurement, and we used it to gain insights into the way it interacts with G4s: the 1:1 stoichiometry of the PNA DOTASQ /G4 association was shown by electrospray ionization mass spectrometry (ESI-MS), and no 2:1 TASQ/G4 complex was detected. The demonstration that this TASQ adopts its closed conformation upon interaction with G4s was provided by NMR (Figure C) .…”