Dissecting Mg2+-RNA interactions using atomistic molecular dynamics

The central dogma of molecular biology summarizes one of the most important mechanisms for the functioning of living organisms, stating that deoxyribonucleic acid (DNA) is transcribed into ribonucleic acid (RNA), which is then translated into proteins. However, it is still not sufficient to capture how important RNA are for cellular life. Nucleic acids are at the core of any living cell on this planet and thus deserve indisputably deserve scientific attention. In particular, RNA molecules are proposed as the key chemical species that ignited the beginning of life on prebiotic earth. Independently of this hypothesis, studying RNA molecules today is essential for numerous applications in life sciences, spanning from drug development to cancer treatment. That being said, in the last half century there have been unprecedented efforts into understanding RNAs and their role in the cell to the utmost detail. RNA is transcribed from DNA and translated into proteins, which then perform an abundance of functions in the cell. On top of that, it can catalyze chemical reactions, regulate gene expression and even carry genetic information which is retrotranscribed into DNA. The outstanding versatility of RNA molecules is due to their unique chemical features, resulting in a very flexible backbone combined with strong interactions between the nucleobases. The balance between canonical base pairs and a multitude of backbone conformations is the main factor for RNA being well structured yet dynamical. On the other hand, RNA folding can only occur in the presence of positively charged particles that compensate the electrostatic repulsion arising from the negatively charged sugar-phosphate backbone, inevitably tying nucleic acids and ions together. Metal ions are instrumental for proper RNA folding and dynamics, while also being crucial cofactors for ribozyme catalysis. Monovalent cations (Na+, K+) are the workhorses compensating the overall negatively charged nucleic acids, while divalent cations are frequently the protagonists of relevant folding events and catalysis. Mg2+ ions, which are the most freely available divalent cations in cells, commonly perform as structural pillars in RNA tertiary structures. Despite the ubiquitous presence of Mg2+ around RNA, the experimental characterization of their interaction is challenging, because Mg2+ do not offer a direct spectroscopic handle for detection and requires high-resolution X-ray crystallography. On top of that, their assignment through X-ray diffraction is difficult, since the Mg2+ is isoelectronic with water and Na+ ions. Therefore, the use of theoretical and computational tools can clearly help reinforce the experimental characterization of Mg2+-RNA interaction and contribute to the most needed dynamical view of these molecules. The results presented in this thesis aim to provide a meaningful description of the interaction between Mg2+ ions and RNA through atomistic molecular dynamics coupled with enhanced sampling techniques. The simulations done in this work were designed to tackle the two most fundamental issues in describing divalent ions interaction with RNA using molecular dynamics. First, the quality and fidelity of the models used, and second the proper sampling of rare events. Through the employment of modified state-of-the-art simulations techniques, I was able to predict Mg2+ binding sites and their correspondent affinities on an RNA duplex. The affinities qualitatively agree with the interaction frequency trends observed in the structural databases (PDB 1 or NDB 2). Furthermore, I evaluated relevant aspects of RNA simulation concerning force field choices for Mg2+ ions, RNA backbone non-bridging oxygens, and water. Lastly, I developed a robust methodological framework that allows for future molecular dynamics simulations aimed to study multiple concurrent binding events associated with high free-energy barriers. Since RNA folding is intrinsically dependent on ionic conditions, I hope that this work will facilitate future research on this important subject.