Unfolding pathways and intermediate states of G-quadruplex nucleic acids: an integrated computational and spectroscopic study

Abstract

G-quadruplex nucleic acids are structurally polymorphic systems whose folding,stability, and function are governed not only by final folded architectures, but also by intermediate states, local interactions, cation coordination, and pathway-dependent rearrangements. This thesis investigates the structural dynamics and spectroscopic signatures of DNA and RNA G-quadruplex systems using an integrated framework combining circular dichroism (CD) and synchrotron radiation circular dichroism spectroscopy (SRCD), NMR-derived structural information, molecular dynamics (MD) simulations, enhanced sampling, free-energy analysis, and TD-DFT/ECD spectralmodelling.

The thesis first examines sequence-encoded frustration in an abridged DNA G-quadruplex system, showing how competing local interactions can direct the formation of stable noncanonical architectures rather than simply destabilizing folding. It then extends this analysis to a higher-order RNA G-quadruplex assembly, where spectroscopic and simulation-supported free-energy interpretation reveal a rugged folding landscape involving local structural persistence, partial rearrangement, and multiple accessible routes. Finally, the unfolding of a G-triplex-related system is investigated through an experiment-guided framework. SRCD identifies a later intermediate-bearing region, which is interpreted through convergent thermodynamic analysis, spectral decomposition, TD-DFT/ECD modelling, and MD simulations as being consistent with a G-hairpin-like ensemble.

Across these systems, the results demonstrate that G-quadruplex folding landscapes are best understood as dynamic and heterogeneous ensembles rather than simple two-state transitions. Methodologically, the thesis provides a practical route for linking spectroscopic observables to structural models in difficult-to-resolve nucleic-acid systems. By distinguishing experimentally anchored conclusions from model-supported structural hypotheses, this work advances the interpretation of G-quadruplex folding, unfolding, and spectral behaviour, and provides a framework applicable to broader studies of nucleic-acid structure, dynamics, ligand recognition, and responsive nucleic-acid architectures.

Thesis is embargoed until 30th September 2027.

Date of Award	Sept 2026
Original language	English
Sponsors	Department for the Economy (Northern Ireland)
Supervisor	Shu-Dong Zhang (Supervisor), Paul McCarron (Supervisor), Mateus Webba Da Silva (Supervisor) & Peter McPherson (Supervisor)