The complexity of even the simplest known life forms makes efforts to synthesize living cells from inanimate components seem like a daunting task. However, recent progress toward the creation of synthetic cells, ranging from simple protocells to artificial cells approaching the complexity of bacteria, suggests that the synthesis of life is now a realistic goal. Protocell research, fueled by advances in the biophysics of primitive membranes and the chemistry of nucleic acid replication, is providing new insights into the origin of cellular life. Parallel efforts to construct more complex artificial cells, incorporating translational machinery and protein enzymes, are providing information about the requirements for protein-based life. We discuss recent advances and remaining challenges in the synthesis of artificial cells, the possibility of creating new forms of life distinct from existing biology, and the promise of this research for gaining a deeper understanding of the nature of living systems.

The nonenzymatic replication of RNA is a potential transitional stage between the prebiotic chemistry of nucleotide synthesis and the canonical RNA world in which RNA enzymes (ribozymes) catalyze replication of the RNA genomes of primordial cells. However, the plausibility of nonenzymatic RNA replication is undercut by the lack of a protocell-compatible chemical system capable of copying RNA templates containing all four nucleotides. We show that short 5′-activated oligonucleotides act as catalysts that accelerate primer extension, and allow for the one-pot copying of mixed sequence RNA templates. The fidelity of the primer extension products resulting from the sequential addition of activated monomers, when catalyzed by activated oligomers, is sufficient to sustain a genome long enough to encode active ribozymes. Finally, by immobilizing the primer and template on a bead and adding individual monomers in sequence, we synthesize a significant part of an active hammerhead ribozyme, forging a link between nonenzymatic polymerization and the RNA world.DOI: http://dx.doi.org/10.7554/eLife.17756.001

The development of a sequence-general nucleic acid copying system is an essential step in the assembly of a synthetic protocell, an autonomously replicating spatially localized chemical system capable of spontaneous Darwinian evolution. Previously described nonenzymatic template-copying experiments have validated the concept of nonenzymatic replication, but have not yet achieved robust, sequence-general polynucleotide replication. The 5′-phosphorimidazolides of the 2′-amino-2′,3′-dideoxyribonucleotides are attractive as potential monomers for such a system because they polymerize by forming 2′→5′ linkages, which are favored in nonenzymatic polymerization reactions using similarly activated ribonucleotides on RNA templates. Furthermore, the 5′-activated 2′-amino nucleotides do not cyclize. We recently described the rapid and efficient nonenzymatic copying of a DNA homopolymer template (dC15) encapsulated within fatty acid vesicles using 2′-amino-2′,3′-dideoxyguanosine−5′-phosphorimidazolide as the activated monomer. However, to realize a true Darwinian system, the template-copying chemistry must be able to copy most sequences and their complements to allow for the transmission of information from generation to generation. Here, we describe the copying of a series of nucleic acid templates using 2′-amino-2′,3′-dideoxynucleotide−5′-phosphorimidazolides. Polymerization reactions proceed rapidly to completion on short homopolymer RNA and LNA templates, which favor an A-type duplex geometry. We show that more efficiently copied sequences are generated by replacing the adenine nucleobase with diaminopurine, and uracil with C5-(1-propynyl)uracil. Finally, we explore the copying of longer, mixed-sequence RNA templates to assess the sequence-general copying ability of 2′-amino-2′,3′-dideoxynucleoside−5′-phosphorimidazolides. Our results are a significant step forward in the realization of a self-replicating genetic polymer compatible with protocell template copying and suggest that N2′→P5′-phosphoramidate DNA may have the potential to function as a self-replicating system.