Taylor-dispersion induced phase separation for the efficient characterisation of protein condensate formation

14 July 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

Biomolecular condensates have emerged as important structures in cellular function and disease, and are thought to form through liquid-liquid phase separation (LLPS). Thorough and efficient in vitro experiments are therefore needed to elucidate the driving forces of protein LLPS and the possibility to modulate it with drugs. Here we present Taylor dispersion induced phase separation (TDIPS), a method to robustly measure condensation phenomena in a commercially available microfluidic platform. It uses only nano-liters of sample, does not require extrinsic fluorescent labels, and is straightforward to implement. We demonstrate TDIPS by screening the phase behaviour of two proteins that form biomolecular condensates \textit{in vivo}, PGL-3 and Ddx4N1. Uniquely accessible to this method, we find an unexpected re-entrant behaviour at very low ionic strength, where LLPS is inhibited for both proteins. TDIPS can also probe the reversibility of assemblies, which was shown for both alpha-synuclein and for Lysozyme, relevant for health and biotechnology, respectively. Finally, we highlight how effective inhibition concentrations and partitioning of LLPS-modifying compounds can be screened efficiently at high throughput.

Keywords

Taylor dispersion
non-equilibrium behaviour
liquid-liquid phase separation
protein
DNA
screening
diffusion

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