Abstract:

Liquid-liquid phase separation (LLPS) has recently been demonstrated as a key mechanism for the

formation of intracellular membraneless organelles, providing novel insights into prebiotic compartment

assembly on early Earth. By creating liquid compartments that mimic the microenvironments found within

organelles and cells, which could term as 'organelloids' or 'celloids,' LLPS presents promising approaches

for the development of carriers and reactors in drug screening and therapeutic applications. In this

presentation, I will discuss my research on the fundamental principles of LLPS and explore the potential

of integrating LLPS with microfluidic high-throughput screening techniques for the construction of the

next-generation drug screening systems.

First, I will demonstrate evaporation-triggered segregative liquid-liquid phase separation (LLPS) within a

single-phase sessile droplet of a polyethylene glycol (PEG) and dextran mixture. I will discuss the kinetic

pathway of phase separation, triggered by the non-uniform evaporation rate and the Marangoni flow-driven

hydrodynamics within the sessile droplet. More importantly, this evaporation-triggered phase-separating

system creates an ideal microenvironment for prebiotic compartmentalization, as evidenced by the

localization and storage of nucleic acids, in vitro transcription, and a three-fold enhancement of ribozyme

activity. Next, I will explore the associative phase separation of RNA oligonucleotides and cationic

peptides, demonstrating that RNA-peptide condensates exhibit tunable material properties across a broad

range due to interactions influenced by RNA folding/unfolding kinetics. These tunable material states can

lead to distinct microenvironments, which further regulate biochemical processes such as RNA aptamer

compartmentalization and RNA cleavage reactions. Lastly, I will discuss the potential of incorporating

LLPS into a microfluidic high-throughput screening platform, which could offer significant advantages

over traditional water-in-oil emulsion-based microfluidic screening systems in various aspects, such as

addressing the long-standing 'off-target' problem. Overall, these findings illustrate how the fundamental

principles of LLPS can be harnessed to create diverse 'organelloids' for different biochemical processes,

offering enormous potential for the next-generation drug screening systems.