Modi, N. | bioRxiv Science

bioRxiv2025

Transient pH changes drive vacuole formation in enzyme-polymer condensates

Intracellular membraneless organelles formed by the phase separation of biomolecules are essential for cellular functioning. These biomolecular condensates often exhibit complex morphologies in response to biological stimuli. In vitro condensate models help elucidate the mechanism of formation and the associated function of these hierarchical assemblies. Here, we use an in vitro model to investigate the formation of hollow internal regions, or vacuoles, within the condensate interior in response to a pH change. Our experimental system is a pH-responsive complex coacervate formed by the anionic glucose oxidase enzyme phase separating with the weak polycation, DEAE-dextran. Fast rates of pH decrease and larger droplet sizes trigger vacuole development within the coacervates. We show that the emergence of vacuoles is a non-equilibrium process caused by the diffusion-limited exchange of condensate components during a fast pH change. We develop a theoretical model that captures how a phase-separating system responds dynamically to changes in system conditions, particularly pH. Our qualitative phase diagram aligns with our experimental results, showing that rapid pH changes shift the phase boundaries, triggering spinodal decomposition and inducing vacuole formation within the condensates. Our pH-sensitive in vitro coacervate model provides a platform to modulate the internal structure of ternary phase separating systems and gain insights into the mechanisms controlling condensate organization in vivo.

biophysics↗

bioRxiv2023

Targeted Phasing of 2-200 Kilobase DNA Fragments with a Short-Read Sequencer and a Single-Tube Linked-Read Library Method

In the human genome, heterozygous sites are genomic positions with different alleles inherited from each parent. On average, there is a heterozygous site every 1-2 kilobases (kb). Resolving whether two alleles in neighboring heterozygous positions are physically linked--that is, phased--is possible with a short-read sequencer if the sequencing library captures long-range information. TELL-Seq is a library preparation method based on millions of barcoded micro-sized beads that enables instrument-free phasing of a whole human genome in a single PCR tube. TELL-Seq incorporates a unique molecular identifier (barcode) to the short reads generated from the same high-molecular-weight (HMW) DNA fragment (known as linked-reads). However, genome-scale TELL-Seq is not cost-effective for applications focusing on a single locus or a few loci. Here, we present an optimized TELL-Seq protocol that enables the cost-effective phasing of enriched loci (targets) of varying sizes, purity levels, and heterozygosity. Targeted TELL-Seq maximizes linked-read efficiency and library yield while minimizing input requirements, fragment collisions on microbeads, and sequencing burden. To validate the targeted protocol, we phased seven 180-200 kb loci enriched by CRISPR/Cas9-mediated excision coupled with pulse-field electrophoresis, four 20 kb loci enriched by CRISPR/Cas9-mediated protection from exonuclease digestion, and six 2-13 kb loci amplified by PCR. The selected targets have clinical and research relevance (BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, PMS2, SCN5A-SCN10A, and PKI3CA). These analyses reveal that targeted TELL-Seq provides a reliable way of phasing allelic variants within targets (2-200 kb in length) with the low cost and high accuracy of short-read sequencing.

genomics↗