Research Projects

The cellular interior presents a highly concentrated milieu for biomacromolecules and solutes, where spatial confinement and restricted solvent availability significantly alter the molecular behaviour. This phenomenon, broadly termed macromolecular crowding (MC),¹ ² is known to reshape the energy landscapes governing nucleic acid structure, folding kinetics, and molecular recognition. In particular, non-canonical nucleic acid structures such as G-quadruplex DNA and RNA exhibit pronounced sensitivity to their microenvironment, leading to altered binding profiles for small molecules and the conformational states of the G-quadruplex DNA (GqDNA) and RNA (GqRNA) structures. Despite their biological significance in genomic stability,³ gene regulation,⁴ and therapeutic targeting,⁵ ⁶ the folding/unfolding mechanisms and ligand-binding kinetics of GqDNA under crowded or dehydrated conditions remain poorly understood. In this project, we propose to use deep eutectic solvents (DESs)⁷ as model environments to mimic cellular crowding. DESs are a new class of green, biodegradable, and tunable liquids formed by mixing two or more components that can interact through hydrogen bonding. By adjusting their composition, DESs can be made to closely resemble the crowded and dehydrated conditions inside cells. These systems offer an environmentally friendly and controllable way to study how nucleic acids behave in non-traditional solvent environments for stability as well as storage. Our goal is to understand how these DES-induced environmental changes affect (1) the ligand binding kinetics to GqDNA/GqRNA, and (2) the conformational heterogeneity of the GqDNA. To study ligand binding kinetics, we will use fluorescence correlation spectroscopy (FCS) at (near) single molecule level, which allows to measure the association (k+) and dissociation (k-) rates of a ligand binding to GqDNA/GqRNA in various DES environments. We plan to use Cresyl Violet (CV) and Berbarine as ligands to study their interactions with the GqDNA/GqRNA. To study conformational changes, we will also explore the single-molecule Forster Resonance Energy Transfer (smFRET). In this technique, DNA molecules will be labelled with two fluorescent dyes, one donor and one acceptor. Changes in the distance between the dyes during folding or unfolding result in measurable changes in energy transfer, allowing us to detect multiple structural states and transitions. We plan to use the Cy3-donor / Cy5-aceptor labelled human telomeric GqDNA sequence for this study. To complement the experimental observations, atomistic molecular dynamics (MD) simulations will be performed in explicit DES environments, enabling direct comparison with spectroscopic results. Furthermore, enhanced sampling techniques, such as well-tempered metadynamics,⁸ will be utilized to reconstruct multidimensional free energy surfaces, identifying metastable states of the ligand binding to GqDNA/GqRNA under DES-induced crowding.