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

Eun Sun Hong 0 1 3 Hye-Joo Yoon 0 1 3 Byungjoo Kim 0 1 2 Yong-Hyeon Yim 0 1 2 Hun-Young So 0 1 2 Seung Koo Shin skshin@postech 0 1 3 0 Address reprint requests to Dr. H.-J. Yoon and Dr. S. K. Shin, Department of Chemistry, Pohang University of Science and Technology , Pohang, Kyungbuk 790-784, Korea 1 Published online April 2, 2010 Received December 14, 2009 Revised March 9, 2010 Accepted March 10, 2010 2 Division of Metrology for Quality Life, Korea Research Institute of Standards and Science , Daejeon, Korea 3 Bio-Nanotechnology Center, Department of Chemistry, Pohang University of Science and Technology , Pohang, Korea The binding sites and consecutive binding constants of alkali metal ions, (M Na , K , Rb , and Cs ), 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. (J Am Soc Mass Spectrom 2010, 21, 1245-1255) 2010 American Society for Mass Spectrometry - 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 ligandnucleic acid interactions. Moreover, ESI-MS has been successfully used in validating the G4 structure of TBA [13], sequencing oligonucleotides [16, 17], and obtaining the IR spectra of G4 DNA [18]. In this report, we generated the 1:1 and 1:2 TBA alkali metal (sodium, potassium, rubidium, and cesium) complex ions by ESI, determined the consecutive binding constants, and carried out sequencing by infrared multiphoton dissociation (IRMPD). To check the structure of TBA in ESI solvent in the presence or absence of alkali metal ions, we performed CD analysis. For the binding constant measurement [15, 19 24], we used the titration method [21, 23, 24]. The relative abundances of metal-bound and unbound Asequence d(GGTTGGTGTGGTTGG) binds and insingle-stranded DNA containing the 15-nucleotide hibits thrombin, thus called thrombin-binding aptamer (TBA) [1]. 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 6]. Some metal ions induce the structural transition to the G4 structure upon binding [710]. 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 TBAmetal 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 TBAmetal (metal potassium, strontium and barium) complexes in the gas TBA in the ESI mass spectra were used to calculate the consecutive binding constants. To determine the metal binding sites, we applied IRMPD [16, 25]. As the efficient absorption of IR photons at 10.6 m by phosphate groups induces phosphodiester backbone cleavage [26 29], the binding sites of metal ions can be directly determined from the fragmentation pattern and the fragment charge distribution. By taking both the consecutive binding constants and the binding sites into consideration, we suggest a specific interaction between alkali metal ions and G4 TBA formed in ESI solvent. Sample Preparation TBA purified by C18-column was purchased from Bioneer (Daejeon, Korea) and used without further purification. Sodium chloride (99.999%), potassium chloride (99.999%), rubidium chloride (99 %), cesium chloride (99.999 %), and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). A stock solution of TBA (100 m) was prepared by dissolving dry powder in ammonium acetate buffer (150 mm, pH 7.0), immersing a sample tube containing TBA in a 95 C oil bath for 10 min, and then cooling it to room temperature. ESI samples were prepared by diluting the stock solution to a 1:10 ratio in a 1:1 (vol/vol) water/isopropanol solution. Both TBA and alkali metal chloride stock solutions were mixed in water and then mixed with isopropanol for ESI. The final solute concentration was 10 m for TBA, 100 m for metal chloride, and 15 mm for ammonium acetate. A 12-Tesla Fourier-transform ion cyclotron resonance (FT-ICR) spectrometer (Varian, Lake Forrest, CA, USA) was used for both titration and IRMPD experiments. This instrument was equipped with a Nanomate-100 chip (Advion, Ithaca, NY, USA) for ESI and a Synrad 48 2 CO2 laser (Mukilteo, WA, USA) for IRMPD as previously described in detail [30]. The ESI mass spectra were taken in negative mode under Nanomate conditions of 1.6-kV applied voltage and 0.2-p.s.i. nitrogen pressure. The ion of interest was mass-selected by ejecting all unwanted ions using stored waveform inverse Fourier transform (SWIFT). Remaining ions in the ICR cell were irradiated with 10.6 m CO2 laser for 300 ms by increasing the laser fluence from 0.56 to 1.25 J cm2. The IR laser beam passed through a BaF2 vacuum window and crossed a cylindrical ICR cell once. ICR transients were acquired in broadband mode with 1.024 M data points at the sampling rate of 2 MHz in the mass ran (...truncated)