Towards the Development of Structure-Based Electrostatic Models Integrating Polyelectrolyte Theories to Investigate Nucleic Acid Folding

Mainan, Avijit (2026) Towards the Development of Structure-Based Electrostatic Models Integrating Polyelectrolyte Theories to Investigate Nucleic Acid Folding. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (Phd thesis of Avijit Mainan (20RS116)) 20RS116.pdf - Submitted Version Restricted to Repository staff only Download (21MB)

Abstract

Nucleic acid folding is governed by a delicate interplay between intrinsic structural interactions and the surrounding ionic environment, with electrostatics playing a central role due to the high charge density of the phosphate backbone. Physiological concentrations of counterions are therefore essential for stabilizing folded nucleic acid structures and enabling functional conformational dynamics. Among nucleic acids, RNA is particularly sensitive to its ionic environment, with Mg²⁺ and K⁺ ions playing a central role in stabilizing RNA tertiary structures. Explicit-solvent molecular dynamics (MD) simulations, together with experimental studies, have established that the RNA ion atmosphere comprises inner-sphere, outer-sphere, and diffuse ions. Despite this detailed understanding, capturing the large-length-scale and long-time-scale phenomena underlying nucleic acid folding remains a major challenge for classical MD simulations. This limitation motivates the development of structure-based electrostatic models that extend the Manning counterion condensation theory of polyelectrolytes to incorporate RNA structural specificity and ion dynamics. In this thesis, structure-based electrostatic frameworks are developed and applied in combination with atomistic MD simulations, free-energy calculations, and Markov state modelling to elucidate ion-driven folding mechanisms in nucleic acids, with a primary focus on RNA. A central component of this work involves extended development and applications of the dynamic counterion condensation (DCC) model that treats the RNA ion atmosphere using a hybrid implicit–explicit representation. The model is rigorously validated through quantitative agreement with a wide range of experimental measurements across diverse RNA systems. Using this framework, the role of outer-sphere hydrated Mg²⁺ ions is examined in several viral RNAs, revealing their active involvement in shaping RNA folding landscapes. One of the studies demonstrates how a dynamic spine of hydrated Mg²⁺ ions modulates minor-groove narrowing and disrupts the stable base triple and quadruple in the Beet Western Yellows Virus pseudoknot. Additionally, this model shows how the ion-induced pierced-lasso topology regulates the functional dynamics of the SARS-CoV-2 frameshifting-stimulatory element, and how Mg²⁺ mediates an equilibrium between ring-open and ring-closed conformations in flaviviral xrRNA through long-range pseudoknot interactions. Building on this foundation, the electrostatic framework is further extended to explicitly capture dynamic exchange between outer-sphere and inner-sphere Mg²⁺ ions through a generalized potential term in the Hamiltonian. This enhanced model accurately predicts specific inner-sphere Mg²⁺ binding sites across diverse RNAs, reproduces experimental observations, and provides mechanistic insight into the distinct roles played by different components of the ion atmosphere in RNA folding and function. The framework is further applied to bridge limitations inherent in high-resolution structural biology techniques. Focusing on the SARS-CoV-2 frameshifting-stimulatory element, this investigates salt-dependent conformational transitions between bent conformations observed in cryo-EM structures and coaxially stacked conformations resolved by X-ray diffraction. Markov state modelling reveals that deletion of the slippery site—a critical regulatory element—substantially alters the native folding pathway mechanism. The transferability of the electrostatic framework is demonstrated by extending it to DNA G-quadruplexes, where excellent agreement with single-molecule FRET experiments validates its applicability beyond RNA systems. Finally, this thesis outlines ongoing efforts toward the development of a three-site-per-nucleotide (3SPN) coarse-grained model by further reducing molecular resolution while retaining essential structural and electrostatic features. Collectively, this work establishes a unified pathway toward the development of structure-based electrostatic models for nucleic acids. The methodologies presented provide advanced computational strategies for investigating how ionic atmospheres govern the folding, dynamics, and function of complex nucleic acid systems across biologically relevant scales.

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