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.

Replication elongates short DNA, reduces sequence bias and develops trimer structure.

The origin of molecular evolution required the replication of short oligonucleotides to form longer polymers. Prebiotically plausible oligonucleotide pools tend to contain more of some nucleobases than others. It has been unclear whether this initial bias persists and how it affects replication. To investigate this, we examined the evolution of 12-mer biased short DNA pools using an enzymatic model system. This allowed us to study the long timescales involved in evolution, since it is not yet possible with currently investigated prebiotic replication chemistries. Our analysis using next-generation sequencing from different time points revealed that the initial nucleotide bias of the pool disappeared in the elongated pool after isothermal replication. In contrast, the nucleotide composition at each position in the elongated sequences remained biased and varied with both position and initial bias. Furthermore, we observed the emergence of highly periodic dimer and trimer motifs in the rapidly elongated sequences. This shift in nucleotide composition and the emergence of structure through templated replication could help explain how biased prebiotic pools could undergo molecular evolution and lead to complex functional nucleic acids.

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.