Skip to main content

Advanced mass spectrometry approaches to reveal nucleic acid folding energy landscapes

Final Report Summary - DNAFOLDIMS (Advanced mass spectrometry approaches to reveal nucleic acid folding energy landscapes)

A molecule’s function is linked to how its structure changes in response to the environment, or upon interacting with other molecules. The object of biophysics is to characterize these different states, their relative stability, and to reveal the mechanisms by which a molecule switches between states. In other words, the aim of biophysics is to characterize the folding energy landscapes. Biophysical sciences rely on analytical methods. The objective of this project was to pushed back the limits of one such analytical methods, mass spectrometry, to study the biophysics of nucleic acids. In essence, mass spectrometry separates species according to their masses, and detects their relative abundances. Here we focused on the interactions between nucleic acids and cations or ligands, with a unique ability to quantify simultaneously all complexes formed in equilibria. Then, by measuring how the equilibria change as a function of the temperature, we reconstruct folding energy landscapes with an unprecedented degree of detail. But in addition to characterizing interaction between molecules (quaternary structure), we also wanted to obtain information on intramolecular forces, and characterize the tertiary structure, i.e. how domains are arranged, and secondary structure, i.e. local repetitive structural motifs.

We first explored what ion mobility spectrometry coupled to mass spectrometry could tell us about the structures of nucleic acids in solution. Before starting this project, we and others were assuming that solution structures were vastly preserved in the gas phase. But we found that this is not true for all structures. Some rigid regions, especially those stabilized by inner cations, are well preserved, but more flexible motifs such as single-stranded loops and double helices got more compact in the gas phase than they were in solution. The contraction, although counterintuitive for multiply charged ions, is favored for relatively low charge states (when most, but not all, phosphate groups are neutralized) when the stabilization provided by new non-native hydrogen bonds overcomes the Coulomb destabilization. It does not mean that local secondary structures are necessarily disrupted, but the tertiary structure rearrangements in the gas phase make the interpretation of ion mobility results less straightforward than usually assumed. This fundamental work, carried out here on nucleic acid model systems, is bound to have a broader impact on mass spectrometry-based structural biology, for a sensible conception of future project and interpretation of ion mobility spectrometry results.

The second methodological advance was to develop a method to directly probe secondary structures in the gas phase. Circular dichroism spectroscopy is a traditional biophysical method to probe secondary structures in solution, and our goal was to make it possible to measure the circular dichroism of ions trapped inside a mass spectrometer. The circular dichroism is the difference of absorption between left- and right-circularly polarized light. In the gas phase, we cannot measure absorption directly, but we use electron photo-detachment to report on the absorption. We found that electron photo-detachment responded differently to different electronic excited states, but this was actually to our advantage for characterizing nucleic acid secondary structures, because the dichroism effect was stronger in the gas phase than in solution. We successfully demonstrated the application of circular dichroism ion spectroscopy for various topologies of G-rich nucleic acids, then leveraged the mass separation to obtain individual circular dichroism spectra of several forms coexisting in a solution mixture. Our demonstration of feasibility thus opens new avenues to study diverse classes of chiral molecules, while leveraging the separation capabilities of contemporary mass spectrometry.