title: Efficient and rapid template-directed nucleic acid copying using 2'-amino-2',3'-dideoxyribonucleoside-5'-phosphorimidazolide monomers.
abstract: 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.
authors.affiliation: 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.
authors.forename: Jason P
authors.initials: JP
authors.lastname: Schrum
authors.forename: Alonso
authors.initials: A
authors.lastname: Ricardo
authors.forename: Mathangi
authors.initials: M
authors.lastname: Krishnamurthy
authors.forename: J Craig
authors.initials: JC
authors.lastname: Blain
authors.forename: Jack W
authors.initials: JW
authors.lastname: Szostak
mesh_terms: D004247:DNA / Q000096:biosynthesis / Q000138:chemical synthesis / Q000235:genetics; D015224:Dideoxynucleosides / Q000737:chemistry* / Q000378:metabolism; D007093:Imidazoles / Q000737:chemistry* / Q000378:metabolism; D009696:Nucleic Acids / Q000096:biosynthesis / Q000138:chemical synthesis* / Q000235:genetics; D012313:RNA / Q000096:biosynthesis / Q000138:chemical synthesis / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013698:Templates, Genetic
chemical_list: D015224:Dideoxynucleosides; D007093:Imidazoles; D009696:Nucleic Acids; D012313:RNA; D004247:DNA