Investigating RNA's Ion Atmosphere, Hydration, and Structural Dynamics Using Classical, Statistical Mechanical and Machine Learning Based Methods

Sarkar, Raju (2025) Investigating RNA's Ion Atmosphere, Hydration, and Structural Dynamics Using Classical, Statistical Mechanical and Machine Learning Based Methods. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Raju Sarkar (19RS113)) 19RS113.pdf - Submitted Version Restricted to Repository staff only Download (13MB)

Abstract

RNA, a highly charged biopolymer composed of negatively charged phosphate groups, overcomes significant electrostatic repulsion to fold into compact, functionally active three-dimensional structures. The stabilization and formation of these structures are strongly influenced by the surrounding counter-ion atmosphere and the organization of hydration layers. Understanding how these environmental factors govern RNA folding and dynamics is crucial to elucidating its structural and functional behavior at the molecular level. In this thesis, a combination of atomistic molecular dynamics simulations, free-energy sampling techniques, and machine learning (ML)-based analysis is employed to investigate various aspects of RNA-ion interactions, hydration patterns, and conformational transitions across multiple scales. These complementary computational strategies provide a unified framework for probing RNA behavior with high spatial and temporal resolution. We first explore the free-energy landscape of Mg²⁺ chelation in a simplistic model system mimicking RNA backbone unit consisting of a Mg²⁺ ion and two dimethyl phosphate (DMP) molecules using well-tempered metadynamics simulations. Our results reveal a diverse set of coordination states, including fully chelated inner-sphere complexes, outer-sphere hexa-hydrated states, and a dynamic ensemble of pre-chelate intermediates. Of particular interest is a solvent-separated interaction mode—referred to as meta-sphere coordination—where Mg²⁺ maintains inner-sphere contact with one phosphate while simultaneously engaging additional phosphates via water-mediated interactions. These intermediate states are found to modulate RNA-ion coordination in larger biological systems, such as the SAM-I aptamer riboswitch. Further, we investigate the impact of two site-specific Mg²⁺ ions—validated by X-ray crystallography and phosphorothioate substitution experiments—on the global conformational dynamics of the SAM-I aptamer. The removal of either ion leads to disruption of a key pseudoknot tertiary interaction, which significantly compromises the riboswitch’s structural integrity and its regulatory function in bacterial systems. To elucidate hydration-mediated effects, we analyze the solvation architecture of three functionally diverse RNAs: SAM-I aptamer, Adenine aptamer, and Flavivirus RNA. These molecules exhibit compact folded topologies with distinct cores measuring 1–1.7 nm in diameter. By spatially decomposing their hydration layers, we observe that the slowest water reorientation dynamics occur in the base-dominated dominated intermediate layers surrounded by key tertiary motifs such as pseudoknots and kink-turns. This finding highlights the intricate coupling between RNA topology and solvent organization. Additionally, the conformational transition of base flipping in HIV TAR RNA is examined through adaptively sampled molecular simulations within a Time-Lagged Independent Component Analysis (TICA) framework. The center-of-mass distance between bases emerges as a robust order parameter, and combined XGBoost regression and interpretable ML model, Shapley Additive exPlanation (SHAP) analysis highlights its pivotal role in dictating transition-state ensembles and free energy barriers, corroborated by mean first passage time results. Collectively, all these findings provide a unified framework linking RNA’s functional dynamics to its ion atmospheres, hydration hierarchies, and underscore advanced computational strategies for exploring multi-scale structural dynamics of RNA at microscopic resolutions.

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