Constrained dynamics of oligonucleotides in the phase-separated droplets

Phase-separated droplets are excellent means of compartmentalizing functional molecules and have been shown as excellent models for protocells. Although complex functions based on oligonucleotides have been studied, we still lack an understanding of how the oligonucleotide dynamics are affected by the condensed internal environment of these droplets. Particularly, we lack high-resolution experimental measurements of the dynamical parameters that control oligonucleotide diffusion inside the phase-separated droplets. In addition, there is no clarity on how these dynamical parameters differ in the charged (coacervates) vs non-charged (aqueous two-phase system, ATPS) environment of these droplets. In this study, using fluorescence correlation spectroscopy (FCS), we demonstrate the constrained dynamics of oligonucleotides inside membraneless phase-separated droplets at an unprecedented resolution. We further compare transport properties at different lengths of oligonucleotides as well as salt concentrations. We observe that among all the parameters the oligonucleotides caging (spatial restriction in the movement) inside the matrix has a considerable impact on the diffusive dynamics. Our study provides a way of unravelling, quantifying and understanding physical parameters governing the polymer transport dynamics inside the phase-separated droplets.