RNA Oligomerisation without Added Catalyst from 2 ',3 '-Cyclic Nucleotides by Drying at Air-Water Interfaces

For the emergence of life, the abiotic synthesis of RNA from its monomers is a central step. We found that in alkaline, drying conditions in bulk and at heated air-water interfaces, 2 ',3 '-cyclic nucleotides oligomerised without additional catalyst, forming up to 10-mers within a day. The oligomerisation proceeded at a pH range of 7-12, at temperatures between 40-80 degrees C and was marginally enhanced by K+ ions. Among the canonical ribonucleotides, cGMP oligomerised most efficiently. Quantification was performed using HPLC coupled to ESI-TOF by fitting the isotope distribution to the mass spectra. Our study suggests a oligomerisation mechanism where cGMP aids the incorporation of the relatively unreactive nucleotides C, A and U. The 2 ',3 '-cyclic ribonucleotides are byproducts of prebiotic phosphorylation, nucleotide syntheses and RNA hydrolysis, indicating direct recycling pathways. The simple reaction condition offers a plausible entry point for RNA to the evolution of life on early Earth

Membrane tension-mediated growth of liposomes : A step closer to synthetic cells

Living cells are highly complex, making it an extremely challenging task to understand how they function. A possible solution is the bottom-up assembly of non-living components and building up life-like features from scratch, i.e., using synthetic cells as a tool to understand the basic characteristics of life. One such chassis for synthetic cells are liposomes, which, like the cell membrane of living cells, are made of phospholipids. As living cells grow, lipids are incorporated into their membrane in order to cope up with the volume increase of the cell. In a similar fashion, a variety of ways are currently being investigated to achieve growth of synthetic cells. Few examples include incorporation of fatty acids from the surrounding environment, reconstituting the enzymes for fatty acid or lipid biosynthesis in the liposome, or by carrying out the synthesis of artificial membrane components through the external addition of precursor molecules. Here, we demonstrate the membrane-tension mediated growth of giant unilamellar vesicles (GUVs) by fusing sub-micrometre-sized feeder vesicles to them. We use a recently developed microfluidic technique, octanol-assisted liposome assembly (OLA), to produce cell-sized (~10 μm) GUVs on-chip. Following the density-based separation of the liposomes from the waste product (1-octanol droplets), we supply small unilamellar vesicles (SUVs, ~30 nm in diameter) which act as a lipid reserve for growth by fusing with the GUVs. The lipids molecules, being very stable in bilayer conformation, require energy to reorient themselves and undergo membrane fusion. We show that increased membrane tension of GUVs can act as a sole driver to carry out multiple fusion events and cause significant growth. By placing a mass population (>1000) of GUVs in a sufficiently hypotonic solution (delta c 3−5 mM), we build up the membrane tension (~10 mN/m) driving multiple SUV-GUV fusionevents, eventually doubling the volume of a part of the population. We probe a variety of lipid compositions, including hybrid (composed of lipids and fatty acids) GUVs and find the growth to be dependent on the lipid composition. Maximum growth is obtained when using a hybrid system, as compared to pure lipids. Our results show the possibility to use a protein-freeminimal system to induce growth in a minimalistic manner and the demonstrated highthroughput microfluidic approach may have useful implications towards realizing an autonomous entity capable of undergoing a continuous growth-division cycle.

Prebiotic synthesis of 3',5'-cyclic adenosine and guanosine monophosphates through carbodiimide-assisted cyclization

3’,5’-Cyclic nucleotides play a fundamental role in modern biochemical processes and have been suggested to have played a central role at the origin of terrestrial life. In this work, we suggest that a formamide-based systems chemistry might account for their availability on the early Earth. In particular, we demonstrate that in a liquid formamide environment at elevated temperatures 3’,5’-cyclic nucleotides are obtained in good yield and selectivity upon intramolecular cyclization of 5’-phosphorylated nucleosides in the presence of carbodiimides.Web of Science2424art. no. e20230051

High-Fidelity Templated Ligation of RNA via 2′,3′-cyclic Phosphate

The templated ligation of oligonucleotides offers a mode of replication in an RNA world. The 2′,3′-cyclic phosphate (>P) is a prebiotically available activation group for RNA and the product of backbone hydrolysis. Using gel electrophoresis and liquid chromatography, we found that the templated ligation of RNA with >P activation proceeds in alkaline (pH 9-11) low-salt aqueous solutions with 1 mM MgCl2 in temperatures ranging from 20 to 25 °C within a few days. Under the optimum conditions of pH 10 and 5 °C, the ligation yielded 40% after 7 days. No additional catalysts were required. In contrast to previous reports, we found an equimolar mixture of 2′-5′ and 3′-5′ linked oligomers in the used conditions. We probed the nucleotide specificity at the ligation site and found that one mutation reduced the ligation yield by 82-92%. We extrapolated these results to a per-nucleotide replication fidelity of 95-98% when ligating 4- to 6-mers. With splinted oligomers, five ligations created a 96 mer strand, demonstrating a possible assembly pathway for long ribozymes. With the low salt requirements, strand separation will be compatible with the ligation conditions using non-equilibrium settings. The findings suggest that templated ligation mediated by 2′,3′-cyclic phosphate in alkaline conditions offer a slow, but precise replication and elongation reaction for RNA on early Earth

Regulating the dynamic folding of a DNA hairpin at the expense of a small, molecular fuel

Molecular machines, such as ATPases or motor proteins, couple the catalysis of a chemical reaction, most commonly hydrolysis of nucleotide triphosphates, to their conformational change. In essence, they continuously convert a chemical fuel to drive their motion. An outstanding goal of nanotechnology remains to synthesize a nanomachine with similar functions, precision, and speed. The field of DNA nan- otechnology has given rise to the engineering precision required for such a device. Simultaneously, the field of systems chemistry developed fast chemical reaction cycles that convert fuel to change the function of molecules. In this work, we thus combined a fast, chemical reaction cycle with the precision of DNA nanotechnology to yield kinetic control over the conformational state of a DNA hairpin. Future work on such systems will result in fast and precise DNA nanodevices

Spatiotemporal control of coacervate formation within liposomes

The understanding of liquid-liquid phase separation is crucial to cell biology and benefits from cell-mimicking in vitro assays. Here, the authors develop a microfluidic platform to study coacervate formation inside liposomes and show the potential of these hybrid systems to create synthetic cells

Spatiotemporal control of coacervate formation within liposomes

Liquid-liquid phase separation (LLPS), especially coacervation, plays a crucial role in cell biology, as it forms numerous membraneless organelles in cells. Coacervates play an indispensable role in regulating intracellular biochemistry, and their dysfunction is associated with several diseases. Understanding of the LLPS dynamics would greatly benefit from controlled in vitro assays that mimic cells. Here, we use a microfluidics-based methodology to form coacervates inside cell-sized (~10 µm) liposomes, allowing control over the dynamics. Protein-pore-mediated permeation of small molecules into liposomes triggers LLPS passively or via active mechanisms like enzymatic polymerization of nucleic acids. We demonstrate sequestration of proteins (FtsZ) and supramolecular assemblies (lipid vesicles), as well as the possibility to host metabolic reactions (β-galactosidase activity) inside coacervates. This coacervate-in-liposome platform provides a versatile tool to understand intracellular phase behavior, and these hybrid systems will allow engineering complex pathways to reconstitute cellular functions and facilitate bottom-up creation of synthetic cells.BN/Cees Dekker LabBN/Marileen Dogterom LabBN/Bionanoscienc

RNA polymerisation without catalyst from 2’,3’-cyclic nucleotides by drying at air-water interfaces

For the emergence of life, the abiotic synthesis of RNA from its monomers is a central step. We found alkaline, uncatalysed drying conditions in bulk and at heated air-water interfaces where 2´,3´-cyclic nucleotides polymerised, forming up to 10-mers within a day. The polymerisation proceeded at a pH range of 7-12 at temperatures between 40-80 °C and was marginally enhanced by K+ ions. Among the canonical ribonucleotides, cGMP polymerised most efficiently. Quantification was performed using HPLC coupled to ESI-TOF by fitting the isotope distribution to the mass spectra. Our study suggests a polymerisation mechanism where cGMP aids the incorporation of the relatively unreactive nucleotides C, A and U. The 2´,3´-cyclic nucleotides are byproducts of prebiotic phosphorylation, nucleotide syntheses and RNA hydrolysis, indicating direct recycling pathways. The simple reaction condition offers a plausible entry point for RNA to the evolution of life on early Earth