Because of its importance for the origin of life, the nonenzymatic copying of RNA templates has been the subject of intense study for several decades. Previous characterizations of template-directed primer extension using 5'-phosphoryl-2-methylimidazole-activated nucleotides (2-MeImpNs) as substrates have assumed a classical in-line nucleophilic substitution mechanism, in which the 3'-hydroxyl of the primer attacks the phosphate of the incoming monomer, displacing the 2-methylimidazole leaving group. However, we have found that the initial rate of primer extension depends on the pH and concentration at which the activated monomer is maintained prior to the primer extension reaction. These and other results suggest an alternative mechanism, in which two monomers react with each other to form an imidazolium-bridged dinucleotide intermediate, which then binds to the template. Subsequent attack of the 3'-hydroxyl of the primer displaces an activated nucleotide as the leaving group and results in extension of the primer by one nucleotide. Analysis of monomer solutions by NMR indicates formation of the proposed imidazolium-bridged dinucleotide in the expected pH-dependent manner. We have used synthetic methods to prepare material that is enriched in this proposed intermediate and show that it is a highly reactive substrate for primer extension. The formation of an imidazolium-bridged dinucleotide intermediate provides a mechanistic interpretation of previously observed catalysis by an activated nucleotide located downstream from the site of primer extension.

The nonenzymatic polymerization of RNA may have enabled copying of functional sequences during the origin of life. Recent progress utilizing 5'-phosphoro-2-aminoimidazole activation has reinvigorated the possibility of using nonenzymatic RNA polymerization for copying arbitrary sequences. However, the reasons why 2-aminoimidazole (AI) is a superior activation group remain unclear. Here we report that the predominant mechanism of polymerization using cytidine-5'-phosphoro-2-aminoimidazolide (Cp*) involves a 2-aminoimidazolium-bridged dinucleotide (Cp*pC) intermediate. To explore the role of this intermediate, we first identify and quantify four reactions involving the synthesis and breakdown of Cp*pC that occur in the absence of the primer-template duplex. We then analyze the dependence of the rate of polymerization on the concentration of the Cp*pC intermediate in the presence and absence of the competitive inhibitor Cp. We also show that the contribution of the monomer Cp* to the polymerization rate is negligible under our primer extension conditions. Finally, we use the experimentally determined rate constants of these reactions to develop a kinetic model that helps explain the changing rate of nonenzymatic RNA polymerization over time. Our model accounts for the concentration of Cp*pC formed by Cp* under primer extension conditions. The model does not completely account for the decline in polymerization rate observed over long times, which indicates that additional important inhibitory processes have not yet been identified. Our results suggest that the superiority of 2-aminoimidazole over the traditional 2-methylimidazole activation is mostly due to the higher level of accumulation of the imidazolium-bridged intermediate under primer extension conditions.

The invariant lineage of Caenorhabditis elegans has powerful potential for quantifying developmental variability in normal and stressed embryos. Previous studies of division timing by automated lineage tracing suggested that variability in cell cycle timing is low in younger embryos, but manual lineage tracing of specific lineages suggested that variability may increase for later divisions. We developed improved automated lineage tracing methods that allow routine lineage tracing through the last round of embryonic cell divisions and we applied these methods to trace the lineage of 18 wild-type embryos. Cell cycle lengths, division axes and cell positions are remarkably consistent among these embryos at all stages, with only slight increases in variability later in development. The resulting quantitative 4-dimensional model of embryogenesis provides a powerful reference dataset to identify defects in mutants or in embryos that have experienced environmental perturbations. We also traced the lineages of embryos imaged at higher temperatures to quantify the decay in developmental robustness under temperature stress. Developmental variability increases modestly at 25°C compared with 22°C and dramatically at 26°C, and we identify homeotic transformations in a subset of embryos grown at 26°C. The deep lineage tracing methods provide a powerful tool for analysis of normal development, gene expression and mutants and we provide a graphical user interface to allow other researchers to explore the average behavior of arbitrary cells in a reference embryo.