Biology subjects

Devaraj, N.

Publications and source records attributed to Devaraj, N..

5 recordsLinked to original sources

bioRxiv2026

A Reaction-Driven Condensate-to-Vesicle Transition Selects, Activates, and Spatially Organizes RNA

Living systems depend on the selective concentration of informational polymers within membrane-bound compartments. Liquid-liquid phase-separated condensates efficiently concentrate biomolecules, whereas membrane-bound vesicles provide persistent compartment boundaries. However, direct chemical mechanisms that couple these organizational states remain largely unexplored. Here we report a reaction-driven pathway linking condensates to RNA-enriched lipid vesicles. Electrostatic interactions between cationic thioesters and RNA drive phase separation into reactive condensates. Reaction with cysteine generates membrane lipids and transforms the condensates into unilamellar bilayer vesicles with near-uniform size distribution. The vesicles encapsulate >90% of the RNA from the initial solution and concentrate it by more than two orders of magnitude to >100 {micro}M. Encapsulation is strongly dependent on RNA length, with short oligonucleotides excluded while longer RNAs are selectively retained. Under spatial gradients of the chemical trigger, sharply defined vesicle populations with distinct RNA compositions emerge. Reaction-driven compartmentalization raises local ribozyme and substrate concentrations above the threshold required for catalytic activity, enabling function from otherwise inactive dilute solutions. These findings establish a mechanism by which chemical reactions generate selective, functional, and spatially organized RNA-enriched membrane-bound compartments from heterogeneous molecular mixtures.

biochemistry↗

bioRxiv2025

De Novo Protocell Membrane Formation Fueled by Primitive Metabolites

Lipid membranes define cell boundaries, acting as gatekeepers for transport and signaling. A central principle in biology is that all cellular membranes descend from a common ancestral membrane, as they cannot be generated in the absence of preexisting lipid structures. It is thus unclear how the first protocell membranes originated from membrane-less precursors. Here we demonstrate the de novo generation of lipid bilayers in the absence of any preexisting membranes, membrane-bound proteins, or lipid nanostructure templates. Using acetate as a two-carbon precursor, lipid tails are constructed by soluble enzymes and spontaneously conjugate to cysteine backbones, forming diacyl lipids that assemble into vesicles. Pore-forming peptides facilitate precursor transport into vesicles, allowing the continuous generation of new lipids. Formation of glycolipid membranes creates compartments that can maintain proton gradients. The de novo formation of membrane bilayers using primitive chemical building blocks may have been an intermediary step between primitive prebiotic biochemistry and the emergence of cellular life.

synthetic biology↗

bioRxiv2023

Encoding extracellular modification of artificial cell membranes using engineered self-translocating proteins

A common method of generating artificial cells is to encapsulate protein expression systems within lipid vesicles. However, to communicate with the external environment, protein translocation across lipid membranes must take place. In living cells, protein transport across membranes is achieved with the aid of complex translocase systems which are difficult to reconstitute into artificial cells. Thus, there is need for simple mechanisms by which proteins can be encoded and expressed inside synthetic compartments yet still be externally displayed. Here we present a genetically encodable membrane functionalization system based on mutants of pore-forming proteins. We show that the membrane translocating loop of -hemolysin can be engineered to translocate functional peptides up to 52 amino acids across lipid membranes. Engineered hemolysins can be used for genetically programming artificial cells to display interacting peptide pairs, enabling their assembly into artificial tissue-like structures capable of signal transduction.

synthetic biology↗

bioRxiv2021

Chemoenzymatic Generation of Phospholipid Membranes Mediated by Type I Fatty Acid Synthase

The de novo formation of lipid membranes from minimal reactive precursors is a major goal in synthetic cell research. In nature, the synthesis of membrane phospholipids is orchestrated by numerous enzymes, including fatty acid synthases and membrane-bound acyltransferases. However, these enzymatic pathways are difficult to fully reproduce in vitro. As such, the reconstitution of phospholipid membrane synthesis from simple metabolic building blocks remains a challenge. Here, we describe a chemoenzymatic strategy for lipid membrane generation that utilizes a soluble bacterial fatty acid synthase (cgFAS I) to synthesize palmitoyl-CoA in situ from acetyl-CoA and malonyl-CoA. The fatty acid derivative spontaneously reacts with a cysteine-modified lysophospholipid by native chemical ligation (NCL), affording a non-canonical amidophospholipid that self-assembles into micron-sized membrane-bound vesicles. To our knowledge, this is the first example of reconstituting phospholipid membrane formation directly from acetyl-CoA and malonyl-CoA precursors. Our results demonstrate that combining the specificity and efficiency of a type I fatty acid synthase with a highly selective bioconjugation reaction provides a biomimetic route for the de novo formation of membrane-bound vesicles.

synthetic biology↗

bioRxiv2020

Heterologous expression of methylxanthine synthesis enzymes in mammalian cells and their use as reporter proteins

This work demonstrates the reconstitution of active methylxanthine synthesis enzymes in human cells and their potential use as inducible reporter enzymes. A variety of plant enzymes involved in caffeine synthesis have been characterized in vitro and several of these methylxanthine synthesis enzymes have been heterologously-expressed in yeast or bacteria. In this work, enzymes from Coffea arabica, Camellia sinensis, and Paullinia cupana have been heterologously-expressed in human cells. We demonstrate that the enzymes tested exhibit similar patterns of activity with a set of xanthine substrates in human cells when compared to previous reports of in vitro activity. We demonstrate that the activity of these enzymes can be used as a reporter for juxtacrine signaling using synNotch-induced expression in the presence of an appropriate substrate. When used in combination with synthetic caffeine receptors, this work has potential for use as an in vivo reporter (e.g. enabling non-invasive monitoring of cell-cell interactions after a cellular transplant) or in synthetic intercellular signaling a methylxanthine, such as caffeine, acting as a synthetic paracrine hormone.

synthetic biology↗