Efficient and Rapid Template-Directed Nucleic Acid Copying Using 2a2-Amino-2a2,3a2-dideoxyribonucleosidea5a2-Phosphorimidazoli de Monomers

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 5a2-phosphorimidazolides of the 2a2-amino-2a2,3a2-dideoxyribonucleotides are attractive as potential monomers for such a system because they polymerize by forming 2a2a5a2 linkages, which are favored in nonenzymatic polymerization reactions using similarly activated ribonucleotides on RNA templates. Furthermore, the 5a2-activated 2a2-amino nucleotides do not cyclize. We recently described the rapid and efficient nonenzymatic copying of a DNA homopolymer template (dC sub(15)) encapsulated within fatty acid vesicles using 2a2-amino-2a2,3a2-dideoxyguanosinea5a2-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 2a2-amino-2a2,3a2-dideoxynucleotidea5a2-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 2a2-amino-2a2,3a2-dideoxynucleosidea5a2-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 N2a2aP5a2-phosphoramidate DNA may have the potential to function as a self-replicating system.