Harnessing multiphasic microdroplets (MPMD) towards nucleic acid engineering

Abstract

Microdroplets have attracted tremendous attentions over a variety of communities in the past decades, among which two representative fields, droplet microfluidics and intracellular liquid-liquid phase separation respectively, have emerged as revolutionary breakthroughs in both science and technologies. As the heart of both droplet microfluidics and intracellular liquid-liquid phase separation, microdroplets formed within two or more co-existing phases, or multiphasic microdroplets, are exhibiting great potentials in many interdisciplinary studies. The present work focuses on harnessing multiphasic microdroplets, with the advanced understanding and manipulation strategies, towards nucleic acid engineering that spans from all-aqueous encapsulation, high-throughput screening to complex coacervation. Upon the understanding and engineering of phase separation dynamics inside an evaporating all-aqueous sessile droplet, an all-aqueous encapsulation strategy of nucleic acids is demonstrated, together with its potentials in prebiotic compartmentalization in origin of life. The kinetic pathway of phase separation triggered by the non-uniform evaporation rate, together with the Marangoni flow-driven hydrodynamics inside the sessile droplet are quantitatively identified. The drying droplets provide a robust mechanism for encapsulation of nucleic acids, as demonstrated by localization and storage of both DNA and RNA, in vitro transcription, as well as a three-fold enhancement of ribozyme activity. Following the all-aqueous encapsulation strategy of nucleic acids, a high throughput droplet microfluidic assisted screening strategy is presented, with its ability in the evolution and selection of new fluorogenic RNA aptamers. In particular, two new aptamers, namely eBroccoli and eCorn respectively, are screened and characterized. Both two new aptamers are able to improve fluorescence intensity and thermal stability upon binding to their fluorophores. Compared with the original aptamers, an enhancement of 1.5 times for eBroccoli and more than 20 times for eCorn in fluorescence intensity is achieved at a high temperature of 42 ℃. The role of RNA structural folding in controlling their complex coacervation of RNA-polycation is also investigated. Liquid-like droplets are formed upon the coacervation between the polycation and RNA with random coils, while solid-like structures appear immediately once the RNA generates a secondary folding structure. The phase transition from liquid to solid or from solid to liquid can be controlled by both charge ratios and salt concentration, providing a promising potential in application such as developing materials with similar composition but distinct morphological and rheological properties. As the concluding remark, a brief review and discussion on the applications of all-aqueous phase separation systems in virus processing is presented, in regarding to the SARS-CoV-2 pandemic. Recent progresses in virus-like particle purification and encapsulation enabled by phase separation are summarized. The potential applications of hydration/dehydration-controlled phase separation in environmental surveillance are discussed. Besides, recent works on the bimolecular condensates formed by nucleocapsid (N) protein of SARS-CoV-2 upon liquid-liquid phase separation are also reviewed. In summary, multiphasic microdroplets towards nucleic acid engineering are presented in this dissertation. Besides the gained scientific understanding, these discoveries also have potentials in various biomedical applications, such as aptamer-based RNA tracking and imaging tools, and developing biocompatible underwater adhesives via RNA-polycation coacervation.