Synthesis of N3′-P5′-linked Phosphoramidate DNA by Nonenzymatic Template-Directed Primer Extension

[Image: see text] A fast and accurate pathway for nonenzymatic RNA replication would simplify models for the emergence of the RNA world from the prebiotic chemistry of the early earth. However, numerous difficulties stand in the way of an experimental demonstration of effective nonenzymatic RNA replication. To gain insight into the necessary properties of potentially self-replicating informational polymers, we have studied several model systems based on amino–sugar nucleotides. Here we describe the synthesis of N3′–P5′-linked phosphoramidate DNA (3′-NP-DNA) by the template-directed polymerization of activated 3′-amino-2′,3′-dideoxyribonucleotides. 3′-NP-DNA is an interesting model because of its very RNA-like A-type duplex conformation and because activated 3′-amino-2′,3′-dideoxyribonucleotides are much more reactive than the corresponding activated ribonucleotides. In contrast to our previous studies with 2′-amino-2′,3′-dideoxyribonucleotides (for which G and C but not A and T exhibit efficient template copying), we have found that all four canonical 3′-amino-2′,3′-dideoxyribonucleotides (G, C, A, and T) polymerize efficiently on RNA templates. RNA templates are generally superior to DNA templates, and oligo-ribo-T templates are superior to oligo-ribo-U templates, which are the least efficient of the RNA homopolymer templates. We have also found that activation of 3′-aminonucleotides with 2-methylimidazole results in a ca. 10-fold higher polymerization rate relative to activation with imidazole, an observation that parallels earlier findings with ribonucleotides. We discuss the implications of our experiments for the possibility of self-replication in the 3′-NP-DNA and RNA systems.