High-Fidelity RNA Copying via 2',3'-Cyclic Phosphate Ligation.

Templated ligation offers an efficient approach to replicate long strands in an RNA world. The 2',3'-cyclic phosphate (>P) is a prebiotically available activation that also forms during RNA hydrolysis. Using gel electrophoresis and high-performance liquid chromatography, we found that the templated ligation of RNA with >P proceeds in simple low-salt aqueous solutions with 1 mM MgCl2 under alkaline pH ranging from 9 to 11 and temperatures from -20 to 25 °C. No additional catalysts were required. In contrast to previous reports, we found an increase in the number of canonical linkages to 50%. The reaction proceeds in a sequence-specific manner, with an experimentally determined ligation fidelity of 82% at the 3' end and 91% at the 5' end of the ligation site. With splinted oligomers, five ligations created a 96-mer strand, demonstrating a pathway for the ribozyme assembly. Due to the low salt requirements, the ligation conditions will be compatible with strand separation. Templated ligation mediated by 2',3'-cyclic phosphate in alkaline conditions therefore offers a performant replication and elongation reaction for RNA on early Earth.

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.

Regulating DNA-Hybridization Using a Chemically Fueled Reaction Cycle.

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 nanotechnology 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 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 out-of-equilibrium DNA nanodevices with precise functions.

Prebiotic Foam Environments to Oligomerize and Accumulate RNA.

When water interacts with porous rocks, its wetting and surface tension properties create air bubbles in large number. To probe their relevance as a setting for the emergence of life, we microfluidically created foams that were stabilized with lipids. A persistent non-equilibrium setting was provided by a thermal gradient. The foam's large surface area triggers capillary flows and wet-dry reactions that accumulate, aggregate and oligomerize RNA, offering a compelling habitat for RNA-based early life as it offers both wet and dry conditions in direct neighborhood. Lipids were screened to stabilize the foams. The prebiotically more probable myristic acid stabilized foams over many hours. The capillary flow created by the evaporation at the water-air interface provided an attractive force for molecule localization and selection for molecule size. For example, self-binding oligonucleotide sequences accumulated and formed micrometer-sized aggregates which were shuttled between gas bubbles. The wet-dry cycles at the foam bubble interfaces triggered a non-enzymatic RNA oligomerization from 2',3'-cyclic CMP and GMP which despite the small dry reaction volume was superior to the corresponding dry reaction. The found characteristics make heated foams an interesting, localized setting for early molecular evolution.

Acid-Catalyzed RNA-Oligomerization from 3',5'-cGMP.

The assembly of ancient informational polymers from nucleotide precursors is the central challenge of life's origin on our planet. Among the possible solutions, dry polymerization of 3',5'-cyclic guanosine monophosphate (3',5'-cGMP) has been proposed as a candidate to create oligonucleotides of 15-20 units in length. However, the reported sensitivity of the reaction to the presence of cations raised questions of whether this chemistry could be relevant in a geological context. The experiments in this study show that the presence of cations is not restrictive as long as the reaction is conducted in an acidic environment, in contrast to previous reports that suggested optimal conditions at pH 9.

Membrane Tension-Mediated Growth of Liposomes.

Recent years have seen a tremendous interest in the bottom-up reconstitution of minimal biomolecular systems, with the ultimate aim of creating an autonomous synthetic cell. One of the universal features of living systems is cell growth, where the cell membrane expands through the incorporation of newly synthesized lipid molecules. Here, the gradual tension-mediated growth of cell-sized (≈10 µm) giant unilamellar vesicles (GUVs) is demonstrated, to which nanometer-sized (≈30 nm) small unilamellar vesicles (SUVs) are provided, that act as a lipid source. By putting tension on the GUV membranes through a transmembrane osmotic pressure, SUV-GUV fusion events are promoted and substantial growth of the GUV is caused, even up to doubling its volume. Thus, experimental evidence is provided that membrane tension alone is sufficient to bring about membrane fusion and growth is demonstrated for both pure phospholipid liposomes and for hybrid vesicles with a mixture of phospholipids and fatty acids. The results show that growth of liposomes can be realized in a protein-free minimal system, which may find useful applications in achieving autonomous synthetic cells that are capable of undergoing a continuous growth-division cycle.

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 (ß-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.