Magnesium ions mitigate metastable states in the regulatory landscape of mRNA elements.

Residing in the 5' untranslated region of the mRNA, the 2'-deoxyguanosine (2'-dG) riboswitch mRNA element adopts an alternative structure upon binding of the 2'-dG molecule, which terminates transcription. RNA conformations are generally strongly affected by positively charged metal ions (especially Mg2+). We have quantitatively explored the combined effect of ligand (2'-dG) and Mg2+ binding on the energy landscape of the aptamer domain of the 2'-dG riboswitch with both explicit solvent all-atom molecular dynamics simulations (99 µsec aggregate sampling for the study) and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) experiments. We show that both ligand and Mg2+ are required for the stabilization of the aptamer domain; however, the two factors act with different modalities. The addition of Mg2+ remodels the energy landscape and reduces its frustration by the formation of additional contacts. In contrast, the binding of 2'-dG eliminates the metastable states by nucleating a compact core for the aptamer domain. Mg2+ ions and ligand binding are required to stabilize the least stable helix, P1 (which needs to unfold to activate the transcription platform), and the riboswitch core formed by the backbone of the P2 and P3 helices. Mg2+ and ligand also facilitate a more compact structure in the three-way junction region.

Oxyaapa: A Picolinate-Based Ligand with Five Oxygen Donors that Strongly Chelates Lanthanides.

Coordination compounds of the lanthanide ions (Ln3+) have important applications in medicine due to their photophysical, magnetic, and nuclear properties. To effectively use the Ln3+ ions for these applications, chelators that stably bind them in vivo are required to prevent toxic side effects that arise from localization of these ions in off-target tissue. In this study, two new picolinate-containing chelators, a heptadentate ligand OxyMepa and a nonadentate ligand Oxyaapa, were prepared, and their coordination chemistries with Ln3+ ions were thoroughly investigated to evaluate their suitability for use in medicine. Protonation constants of these chelators and stability constants for their Ln3+ complexes were evaluated. Both ligands exhibit a thermodynamic preference for small Ln3+ ions. The log KLuL = 12.21 and 21.49 for OxyMepa and Oxyaapa, respectively, indicating that the nonadentate Oxyaapa forms complexes of significantly higher stability than the heptadentate OxyMepa. X-ray crystal structures of the Lu3+ complexes were obtained, revealing that Oxyaapa saturates the coordination sphere of Lu3+, whereas OxyMepa leaves an additional open coordination site for a bound water ligand. Solution structural studies carried out with NMR spectroscopy revealed the presence of two possible conformations for these ligands upon Ln3+ binding. Density functional theory (DFT) calculations were applied to probe the geometries and energies of these conformations. Energy differences obtained by DFT are small but consistent with experimental data. The photophysical properties of the Eu3+ and Tb3+ complexes were characterized, revealing modest photoluminescent quantum yields of <2%. Luminescence lifetime measurements were carried out in H2O and D2O, showing that the Eu3+ and Tb3+ complexes of OxyMepa have two inner-sphere water ligands, whereas the Eu3+ and Tb3+ complexes of Oxyaapa have zero. Lastly, variable-temperature 17O NMR spectroscopy was performed for the Gd-OxyMepa complex to determine its water exchange rate constant of kex298 = (2.8 ± 0.1) × 106 s-1. Collectively, this comprehensive characterization of these Ln3+ chelators provides valuable insight for their potential use in medicine and garners additional understanding of ligand design strategies.