Research Projects

Liquid-liquid phase separation (LLPS) in cells plays a crucial role in the spatiotemporal control of several biochemical processes. Furthermore, several of the sub-cellular LLPS droplets have been associated with pathological conditions, such as viral infections and neurodegenerative disorders. These phase-separated droplets are efficient in partitioning a variety of functional biomolecules, thereby increasing their local concentration. Nevertheless, the internal environment of these droplets is very condensed and crowded, which can drastically affect the dynamics of encapsulated biomolecules and consequently perturb their function. For instance, in the case of ligand binding, phase separation can increase the local concentration of both the ligand and its cognate target, affecting the binding efficiency. Although several studies report the feasibility of ligand binding and other biomolecular functions in crowded environments, we lack a quantitative understanding of the binding dynamics of functional RNA and ligands, particularly in the context of LLPS and at a single-molecule level. The partitioning and diffusion of biomolecules in the LLPS droplets are determined by their size, structural characteristics, and net charge. Thus, in addition to quantitative, the measurements for deciphering the effect of the LLPS microenvironment on the binding-unbinding kinetics also need to be very sensitive and high-resolution. In the proposed project, we aim to decipher the effect of the LLPS microenvironment on the binding-unbinding kinetics of RNA ligand complexes at a single-molecule level. To achieve this, we will employ a single molecule technique, dual-color fluorescence cross-correlation spectroscopy (dcFCCS), which measures time correlations of intensity fluctuations of diffusing biomolecules. The measurements are done at µsec time scales and nm length scales, thereby, the dynamics of the biomolecules can be quantified at an unprecedented spatiotemporal resolution. Here both the ligand and its cognate target will be labelled with orthogonal fluorophores, so their fluorescence intensity fluctuations will be cross-correlated if they interact. Thus, dynamical information on the binding (when the two signals are correlated) and unbinding (when signals are not correlated) of the ligand to its cognate RNA can be deduced by measuring the temporal cross-correlations. For the proposed project, we will use an RNA mimic of green fluorescent protein (GFP), Spinach aptamer, as a model system. This aptamer has been developed for live-cell mRNA imaging and has a conserved G-quadruplex structure (rG4) at its core, responsible for the GFP chromophore binding and fluorescence. Quantifying RNA-ligand binding kinetics in LLPS will not only allow us to identify the factors governing the binding dynamics in the condensed environment but will also lay the foundation for future endeavours to screen small molecules targeting the RNAs in pathological LLPS.