Structures of the kinetically trapped i-motif DNA intermediates

Abstract

In the present work, the conformational dynamics and folding pathways of i-motif DNA were studied in solution and in the gas-phase as a function of the solution pH conditions using circular dichroism (CD), photoacoustic calorimetry analysis (PAC), trapped ion mobility spectrometry - mass spectrometry (TIMS-MS), and molecular dynamics (MD). Solution studies showed at thermodynamic equilibrium the existence of a two-state folding mechanism, whereas during the pH = 7.0 → 4.5 transition a fast and slow phase (ΔHfast+ ΔHslow = 43 ± 7 kcal mol−1) with a volume change associated with the formation of hemiprotonated cytosine base pairs and concomitant collapse of the i-motif oligonucleotide into a compact conformation were observed. TIMS-MS experiments showed that gas-phase, kinetically trapped i-motif DNA intermediates produced by nanoESI are preserved, with relative abundances depending on the solution pH conditions. In particular, a folded i-motif DNA structure was observed in nanoESI-TIMS-MS for low charge states in both positive and negative ion mode (e.g., z = +/− 3 to +/−5) at low pH conditions. As solution pH increases, the cytosine deprotonation leads to the loss of cytosine-cytosine+ (C•CH+) base pairing in the CCC strands and in those conditions we observe partially unfolded i-motif DNA conformations in nanoESI-TIMS-MS for higher charge states (e.g., z = − 6 to −9). Collisional induced activation prior TIMS-MS showed the existence of multiple local free energy minima, associated with the i-motif DNA unfolding at z = −6 charge state. For the first time, candidate gas-phase structures are proposed based on mobility measurements of the i-motif DNA unfolding pathway. Moreover, the inspection of partially unfolded i-motif DNA structures (z = −7 and z = −8 charge states) showed that the presence of inner cations may or may not induce conformational changes in the gas-phase. For example, incorporation of ammonium adducts does not lead to major conformational changes while sodium adducts may lead to the formation of sodium mediated bonds between two negatively charged sides inducing the stabilization towards more compact structures in new local, free energy minima in the gas-phase.

Native Ion Mobility Mass Spectrometry: When Gas-Phase Ion Structures Depend on the Electrospray Charging Process

“…However, the CCS profile of the unfolded structure is very sensitive to the NH4OAc concentration, even though the solution NMR spectra are the same: at 15 mM NH4OAc, the 6-ion conformations are more compact; at 100 mM NH4OAc, only an extended form (CCS = 897 Ų) is observed. Note that the previous study Fernandez-Lima and collaborators [34], who found no marked effect of the pH on the ion mobility of 21C, had been carried out in 10 mM NH4OAc, so our results are consistent with theirs.…”

“…We showed previously by IRMPD ion spectroscopy that electrosprayed i-motif structures preserved the C-H + -C base pairs, at least for low charge states (4-to 6-) [33]. However, Fernandez-Lima and collaborators had found no difference in ion mobility profiles of the human telomeric i-motif sequence d(CCCTAA)3CCC in 10 mM NH4OAc as a function of the pH [34], casting doubt on the potential utility of ESI-IMS-MS to study i-motif solution folding. Here, we show that the ion mobility spectra depend strongly on the electrolyte (NH4OAc) concentration used for electrospray, and we show the importance of control experiments (different pH, NH4OAc concentrations, activation energies, and experiments with control sequences) to infer information on the solution structures from ion mobility measurements.…”

“…Given that these mechanisms should be general, why are electrospray effects (electrolyte concentration effects) so prominent for nucleic acids, but remained under the radar for [55]. Note that in positive mode, nucleic acids can never carry as many charges as in the negative mode [34], even in the presence of supercharging additives [45]. The explanation is that they have no reason to sit on the surface of a positively charged droplet and thus no chance to undergo CEM.…”

Magnifying ion mobility spectrometry–mass spectrometry measurements for biomolecular structure studies

“…In separate but related studies, Garabedian et . al and coworkers used photoacoustic calorimetry analysis (PAC) with trapped ion mobility spectrometry (TIMS)-MS to study structural transformations of i-motif DNA resulting from pH changes [59]. Boschmans et .…”

Analysis of Photoirradiated Water Accommodated Fractions of Crude Oils Using Tandem TIMS and FT-ICR MS

“…83–92 More recently, with the development of trapped ion mobility spectrometry (TIMS), 93–95 several reports have shown the potential of TIMS-MS to decouple the mobility (K) separation from the MS analysis time for fast, gas-phase separation and for molecular structural elucidation. 51, 93, 96–117 In particular, the advantages of TIMS over traditional IMS analyzers has been shown for fast screening 96 and targeted 79 analysis of molecular ions from complex chemical mixtures; the study of isomerization kinetics of small molecules, 98 peptides, DNA, proteins and their complexes in the absence of the bulk solvent; 99–103 the influence of the collision partner on the molecular structure; 104 and the factors that affect molecular-adduct complex lifetime and stability during TIMS measurements. 105 A significant feature to note is the high resolving power of TIMS analyzers (R TIMS up to 400 107 ) and accuracy in measuring ion-neutral collision cross section (CCS, <0.6% error).…”