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 (dC15) 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.

Templating efficiency of naked DNA

“…We speculate that the reactivity of amino-terminal primers and OAt ester of dNMPs is not critical for nucleic acid-based selfreplication (21). Primer extension with natural, hydroxy-terminated primers and dNTPs may be induced by the deprotonation of the 3′-hydroxy group (producing a very strongly nucleophilic oxyanion) or metal binding to the leaving group, or both.…”

“…Copying is faster when the primer terminus is an amine, particularly when the amine is at the 2′ position (19) or part of an alternative structure (20)(21)(22). Successful extensions on mixed sequences have been achieved with amines (20,21), but only when the template was RNA or a close analog that favors A-type helices, and when replacements for uracil and adenine were used.…”

“…Successful extensions on mixed sequences have been achieved with amines (20,21), but only when the template was RNA or a close analog that favors A-type helices, and when replacements for uracil and adenine were used. This leaves open the question how good a template DNA is for primer extension, and thus replication.…”

Fast and accurate nonenzymatic copying of an RNA-like synthetic genetic polymer

“…Obviating the requirement for a polymerase greatly simplifies the task of creating and assembling the components of an artificial cell, and thus of constructing simple living systems from inanimate materials. We and others have therefore explored the synthesis of a variety of phosphoramidatelinked nucleic acids, their corresponding amino-sugar monomers, and the characterization of nonenzymatic template-directed primer extension reactions in these systems (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Among these systems, we have examined 2′-amino versions of the acyclic glycerol nucleic acid (5), 2′-amino-2′,3′-dideoxyribonucleic acid (4,6), and 3′-amino-2′,3′-dideoxyribonucleic acid (7).…”

“…Indeed, we were only able to demonstrate nonenzymatic template copying after the synthesis of activated dinucleotides. In an effort to avoid the problems associated with monomer cyclization, we turned to the 2′-amino-2′,3′-dideoxyribonucleotides, which polymerize into 2′-5′ linked phosphoramidate DNA (6). These monomers do not cyclize, as the cyclic sugar keeps the amine nucleophile away from the activated phosphate electrophile.…”