Nonenzymatic Template-Directed Synthesis of Mixed-Sequence 3′-NP-DNA up to 25 Nucleotides Long Inside Model Protocells

[Image: see text] Efficiently copying mixed-sequence oligonucleotide templates nonenzymatically is a long-standing problem both with respect to the origin of life, and with regard to bottom up efforts to synthesize artificial living systems. Here we report an efficient and sequence-general nonenzymatic process in which RNA templates direct the synthesis of a complementary strand composed of N3′→P5′ phosphoramidate DNA (3′-NP-DNA) using 3′-amino-2′,3′-dideoxyribonucleotides activated with 2-aminoimidazole. Using only the four canonical nucleobases (A, G, C, and T) of modern DNA, we demonstrate the chemical copying of a variety of mixed-sequence RNA templates, both in solution and within model protocells, into complementary 3′-NP-DNA strands. Templates up to 25 nucleotides long were chemically transcribed with an average stepwise yield of 96–97%. The nonenzymatic template-directed generation of primer extension products long enough to encode active ribozymes and/or aptamers inside model protocells suggests possible routes to the synthesis of evolving cellular systems.