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.

Sequence self-selection by cyclic phase separation.

The emergence of functional oligonucleotides on early Earth required a molecular selection mechanism to screen for specific sequences with prebiotic functions. Cyclic processes such as daily temperature oscillations were ubiquitous in this environment and could trigger oligonucleotide phase separation. Here, we propose sequence selection based on phase separation cycles realized through sedimentation in a system subjected to the feeding of oligonucleotides. Using theory and experiments with DNA, we show sequence-specific enrichment in the sedimented dense phase, in particular of short 22-mer DNA sequences. The underlying mechanism selects for complementarity, as it enriches sequences that tightly interact in the dense phase through base-pairing. Our mechanism also enables initially weakly biased pools to enhance their sequence bias or to replace the previously most abundant sequences as the cycles progress. Our findings provide an example of a selection mechanism that may have eased screening for auto-catalytic self-replicating oligonucleotides.

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.

Continuous nonenzymatic cross-replication of DNA strands with in situ activated DNA oligonucleotides.

Continuous enzyme-free replication of oligonucleotides is central for open-ended evolution experiments that mimic the origin of life. Here, we studied a reaction system, whereby two 24mer DNA templates cross-catalyzed each other's synthesis from four 12mer DNA fragments, two of which were in situ activated with the condensing agent 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC). We circumvented the problem of product inhibition by melting the stable product duplexes for their reuse as templates in the following ligation step. The system reproduced itself through ligation/melting cycles and survived exponential dilution. We quantified EDC-induced side reactions in a detailed kinetic model. The model allowed us to analyze the effects of various reaction rates on the system's kinetics and confirmed maximal replication under the chosen conditions. The presented system enables us to study nonenzymatic open-ended evolution experiments starting from diverse sequence pools.

Heated gas bubbles enrich, crystallize, dry, phosphorylate and encapsulate prebiotic molecules.

Non-equilibrium conditions must have been crucial for the assembly of the first informational polymers of early life, by supporting their formation and continuous enrichment in a long-lasting environment. Here, we explore how gas bubbles in water subjected to a thermal gradient, a likely scenario within crustal mafic rocks on the early Earth, drive a complex, continuous enrichment of prebiotic molecules. RNA precursors, monomers, active ribozymes, oligonucleotides and lipids are shown to (1) cycle between dry and wet states, enabling the central step of RNA phosphorylation, (2) accumulate at the gas-water interface to drastically increase ribozymatic activity, (3) condense into hydrogels, (4) form pure crystals and (5) encapsulate into protecting vesicle aggregates that subsequently undergo fission. These effects occur within less than 30 min. The findings unite, in one location, the physical conditions that were crucial for the chemical emergence of biopolymers. They suggest that heated microbubbles could have hosted the first cycles of molecular evolution.