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J Am Chem Soc. 2009 Oct 14;131(40):14560-70. doi: 10.1021/ja906557v.

Efficient and rapid template-directed nucleic acid copying using 2'-amino-2',3'-dideoxyribonucleoside-5'-phosphorimidazolide monomers.

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Howard Hughes Medical Institute and Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Harvard Medical School, Boston, Massachusetts 02114, USA.


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 (dC(15)) 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.

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