Mass spectrometric studies of alkali metal ion binding on thrombin-binding aptamer DNA

Abstract

The binding sites and consecutive binding constants of alkali metal ions, (M ϩ ϭ Na ϩ , K ϩ , Rb ϩ , and Cs ϩ ), to thrombin-binding aptamer (TBA) DNA were studied by Fouriertransform ion cyclotron resonance spectrometry. TBA-metal complexes were produced by electrospray ionization (ESI) and the ions of interest were mass-selected for further characterization. The structural motif of TBA in an ESI solution was checked by circular dichroism. The metal-binding constants and sites were determined by the titration method and infrared multiphoton dissociation (IRMPD), respectively. The binding constant of potassium is 5-8 times greater than those of other alkali metal ions, and the potassium binding site is different from other metal binding sites. In the 1:1 TBA-metal complex, potassium is coordinated between the bottom G-quartet and two adjacent TT loops of TBA. In the 1:2 TBA-metal complex, the second potassium ion binds at the TGT loop of TBA, which is in line with the antiparallel G-quadruplex structure of TBA. On the other hand, other alkali metal ions bind at the lateral TGT loop in both 1:1 and 1:2 complexes, presumably due to the formation of ion-pair adducts. IRMPD studies of the binding sites in combination with measurements of the consecutive binding constants help elucidate the binding modes of alkali metal ions on DNA aptamer at the molecular level. . The active form of TBA adopts a chair-type intramolecular G-quadruplex (G4), where the two G-tetrads are interconnected through the lateral TT and TGT loops in antiparallel conformation [2][3][4][5][6]. Some metal ions induce the structural transition to the G4 structure upon binding [7][8][9][10]. The binding modes of metal ions on the G4 structure are of fundamental interest in understanding their specific and/or nonspecific interactions. Various methods have been applied to the studies of binding modes and structures of TBA-metal complexes. NMR experiments have shown that potassium induces the chair-type G-quadruplex formation, but sodium does not [11]. Circular dichroism (CD) and UV absorption studies have shown that potassium, strontium, and barium stabilize the G4 structure in solution [12]. Recently, electrospray ionization mass spectrometry (ESI-MS) has confirmed the unimolecular G4 structure of TBA-metal (metal ϭ potassium, strontium and barium) complexes in the gas phase by H/D exchange [13]. Here, we studied the binding modes of alkali metal ions on TBA and determined the consecutive binding constants to unravel the specific and/or non-specific interactions between alkali metal ions and G4 TBA.Most of the structural studies carried out in solution deal with an ensemble of dynamic structures present under ambient conditions. To more firmly establish the binding mode, we need to employ a molecular probe that is selective toward the specific ions of interest. ESI-MS offers a mass-specific means to bring the complex ions present in solution into the gas-phase [14,15] for the studies of noncovalent ligand-nucleic acid interactions. Moreover, ESI-MS h...

Cation binding to 15-TBA quadruplex DNA is a multiple-pathway cation-dependent process

“…However, the simulations predicted formation of 1:1 complex between 15-TBA and the ion, contrasting some studies suggesting 1:2 complex with vacant internal cavity of the quadruplex (46,47,70), with the ions located in the TGT and TT loop regions. Here, we address this issue with a combination of explicit solvent MD (30 new individual trajectories with aggregate length of 4 μs), hybrid QM/MM approach and ITC measurements.…”

Ammonium Ion Binding to DNA G-Quadruplexes: Do Electrospray Mass Spectra Faithfully Reflect the Solution-Phase Species?

“…The peak corresponding to two ammonium ions bound is the base peak for charge state 3-. The existence of a second cation binding site for the TBA in solution is currently controversial [30,[37][38][39]. Our mass spectrometry results clearly show the preservation of an additional specific ammonium cation by the TBA in addition to the one likely bound between the G-quartets, suggesting that a fraction of the population in solution possessed this additional cation.…”

“…, has been studied by ESI-MS previously [28][29][30], but the detection of ammonium ion binding to TBA has not yet been reported. For the latter three sequences, the ammonium ion binding constants have been determined previously by NMR [25,27], so the theoretical distribution of ammonium ions captured between the G-quartets of the quadruplexes in solution can be calculated (Table 1).…”

Potential uses of G-quadruplex-forming aptamers

“…However, the localization of ions in the quadruplex and the stoichiometry of binding are not yet fully clear and, in certain cases, controversial results have been reported. Researches using NMR (Marathias and Bolton 2000), infrared multiphoton dissociation (IRMPD) and CD methods (Hong et al 2010) to study the TBA sequence have shown that the most likely form of 1:2 (TBA to K + ) stoichiometry is that which is depicted schematically in Fig. 1C.…”