Examining DNA Structures with In-droplet Hydrogen/Deuterium Exchange Mass Spectrometry

Abstract

Capillary vibrating sharp-edge spray ionization (cVSSI) combined with hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has been utilized to characterize different solution-phase DNA conformers including DNA G-quadruplex topologies as well as triplex DNA and duplex DNA. In general, G-quadruplex DNA shows a wide range of protection of hydrogens extending from ~12% to ~21% deuterium incorporation. Additionally, the DNA sequences selected to represent parallel, antiparallel, and hybrid G-quadruplex topologies exhibit slight differences in deuterium uptake levels which appear to loosely relate to overall conformer stability. Notably, the exchange level for one of the hybrid sequence sub topologies of G-quadruplex DNA (24 TTG) is significantly different (compared with the others studied here) despite the DNA sequences being highly comparable. For the quadruplex-forming sequences, correlation analysis suggests protection of base hydrogens involved in tetrad hydrogen bonding. For duplex DNA ~19% deuterium incorporation is observed while only ~16% is observed for triplex DNA. This increased protection of hydrogens may be due to the added backbone scaffolding and Hoogsteen base pairing of the latter species. These experiments lay the groundwork for future studies aimed at determining the structural source of this protection as well as the applicability of the approach for ascertaining different oligonucleotide folds, co-existing conformations, and/or overall conformer flexibility.

Figure 1.

Schematic diagram of the dual cVSSI setup to study DNA structures. Required component parts to perform in-droplet HDX-MS experiments are labeled. The diagram is not drawn to scale.

Figure 2.

CD spectra of representative G-quadruplex topologies. Shown are spectra for Nmyc (parallel, A), 22GT (antiparallel, B) and hybrid-1 (24TTG, C). All DNA quadruplex solutions are maintained under the same conditions used for the MS experiments (i.e., 10 uM DNA concentration in 100 mM TEAA and 1 mM KCl).

Figure 3.

Mass spectra for representative analytes. The green colored isotopic distributions (A, C, and E) represent conditions before deuterium incorporation. The red colored distributions (B, D, and F) represent conditions for deuterium incorporation. Results are shown for the internal standard dT₆ (A, B), 24TTG (C, D) and 23TTG (E, F) ions. Here the zoomed regions represent the [M-2H]²⁻ and [M-7H+2K⁺]⁵⁻ ions for the internal standard and G-quadruplex species, respectively. The G-quadruplex ions are those containing two K+ adducts.

Figure 4.

Correlation plots showing deuterium uptake dependence. Panel A shows the correlation for total experimental deuterium uptake for unfolded structures versus their corresponding folded G-quadruplex species. For panels B, C, D, E, and F, the x-axis represents the number of exchangeable hydrogens and the y-axis represents the experimental deuterium uptake. For unfolded molecules, panels B and C show the relationship of deuterium uptake with all of the exchangeable hydrogens and those on neutral phosphates only, respectively. Panel D, E, and F show the results obtained for the folded G-quadruplex species. For panels D and E, the exchangeable hydrogens are all heteroatom hydrogens and all such hydrogens while rejecting the outlier (24TTG), respectively. Panel E shows the relationship when the exchangeable hydrogens are limited to neutral phosphate sites.

Figure 5.

Isotopic distributions of 18-mer DNA species and the internal standard. Green colored mass spectra (Panels A, C, E, and G) show results prior to deuterium incorporation. The red colored spectra (Panels B, D, F, and H) represent data obtained upon deuterium incorporation. Results are shown for dT₆ (Panels A and B), 18GAA (Panels C and D), 18TTC (Panels E and F), and 18GAA.TTC triplex DNA (Panel G and H) ions. Here the zoomed regions represent the [M-2H] ²⁻ ions for the internal standard, [M-4H] ⁴⁻ ions for the ss 18GAA and ss 18TTC strands and the [M-7H] ⁷⁻ ions for the 18 GAA.TTC triplex DNA ions. Panels A, C, E, G show results obtained before the introduction of D₂O reagent and panels B, D, F and H show those after the introduction of D₂O.