Exploring RNA-Ion Coordination Modes and Their Impacts on RNA Structure Using Various Computer Simulation Methods

Jaiswar, Akhilesh (2025) Exploring RNA-Ion Coordination Modes and Their Impacts on RNA Structure Using Various Computer Simulation Methods. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Akhilesh Jaiswar (19RS114)) 19RS114.pdf - Submitted Version Restricted to Repository staff only Download (8MB)

Abstract

RNA is a biopolymer consisting of a negatively charged phosphate backbone. It often folds into compact tertiary structures and performs various biological functions, necessitating counterions to mitigate electrostatic repulsion. This thesis explores the microscopic interactions between counterions and RNA using atomistic molecular dynamics simulations, free-energy calculations, and enhanced sampling methods such as well-tempered metadynamics. The thesis includes five interconnected chapters: Chapter 1 provides an introduction to RNA structure, the hierarchy of RNA folding, ion preferences, and the critical role of magnesium ion in RNA folding. Chapter 2 discusses the challenges of simulating RNA, particularly force-field-related issues with magnesium-RNA interactions. This chapter also outlines the theoretical foundations and computational methodologies employed in this work. This includes molecular dynamics simulations, thermodynamic free energy analysis methods and force-field parameters for RNA-ion interactions. To address slow magnesium-phosphate exchange kinetics, in Chapter 3, we quantify the exchange barrier by performing umbrella sampling and compare various magnesium force-field parameters against experimental barrier values. Chapter 3 also characterizes different counterion coordination modes in RNA ion environment. Through atomistic simulations of various RNA systems ranging from a simple pseudoknot (PK) in Beet Western Yellow Virus to complex flaviviral RNA PKs, we identify three distinct peaks in the radial distribution function (RDF). Based on these peaks, we classify RNA-ion coordination into two primary categories: (i) Direct (inner-sphere) coordination – where ions directly coordinate with a single or multiple phosphate groups, the latter referred to as ‘chelation.’ (ii) Indirect (solvent-separated) coordination – further subdivided into single-solvent-separated (outer-sphere) and multi-solvent-separated modes. To characterize thermodynamics of ion-chelation, we developed a simple model system with two dimethyl phosphate (DMP) molecules directly coordinating a single magnesium ion. Using well-tempered metadynamics, we mapped the free energy landscape of chelation, revealing four distinct states: (i) Chelated, (ii) Pre-Chelate 1 (PC1), (iii) Pre-Chelate 2 (PC2), and (iv) Outer sphere. In Pre-Chelate states, magnesium simultaneously maintains direct coordination with one phosphate and solvent-separated interactions with multiple phosphates—a hybrid behavior we term "meta-sphere coordination." Additionally, we observe that local phosphate oxygen atoms exhibit "dangling" behavior, allowing transient water molecule inclusion. The existence of Pre-Chelate complexes in complex RNA systems, such as the SAM-I aptamer RNA is observed. Free-energy calculations of magnesium chelation in this RNA system reveal five distinct states: (i) Chelated, three pre-chelate complexes as (ii) PC1, (iii) PC2, (iv) PC3, and (v) Outer-sphere coordination. The local phosphate oxygen’s "dangling" behavior as observed in the DMP model system is also present in the SAM-I RNA aptamer. Chapter 4 explores the functional impact of chelation on the structure/function of SAM-I RNA aptamer, a critical non-coding RNA that regulates bacterial transcription. X-ray crystallography has identified two site-specific magnesium ions: (i) core-chelated and (ii) exposed-chelated. However, in a cellular environment, additional inner- and outer-sphere magnesium ions may exist. Phosphorothioate interference mapping experiments confirm the presence of various magnesium binding sites in the simulated structures. Our simulations reveal that both core-chelated and exposed-chelated magnesium ions are essential for maintaining the tertiary pseudoknot interaction of the SAM-I riboswitch. We mechanistically demonstrate how the absence of any of these chelated magnesium ions leads to the disruption of functional tertiary interactions. Chapter 5 concludes by integrating insights from the preceding chapters to establish a unified framework that connects RNA’s structural and functional dynamics to its ionic environment. The chapter also highlights the power of advanced computational approaches in uncovering atomistic-level insights into RNA behaviour and outlines future directions that stem from the findings of this thesis.

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