Schrum Jason P, Ricardo Alonso, Krishnamurthy Mathangi, Blain J Craig, Szostak Jack W
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.
J Am Chem Soc. 2009 Oct 14;131(40):14560-70. doi: 10.1021/ja906557v.
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.
开发一种序列通用的核酸复制系统是合成原始细胞组装过程中的关键一步，原始细胞是一种能够自发进行达尔文式进化的自主复制、空间定位的化学系统。先前描述的非酶模板复制实验验证了非酶复制的概念，但尚未实现强大的、序列通用的多核苷酸复制。2'-氨基-2',3'-二脱氧核糖核苷酸的5'-磷酰咪唑化物作为该系统潜在的单体很有吸引力，因为它们通过形成2'→5'键进行聚合，在使用RNA模板上类似活化的核糖核苷酸的非酶聚合反应中这种键更受青睐。此外，5'-活化的2'-氨基核苷酸不会环化。我们最近描述了使用2'-氨基-2',3'-二脱氧鸟苷-5'-磷酰咪唑化物作为活化单体，对包裹在脂肪酸囊泡中的DNA同聚物模板（dC(15)）进行快速高效的非酶复制。然而，要实现一个真正的达尔文系统，模板复制化学必须能够复制大多数序列及其互补序列，以便信息代代相传。在这里，我们描述了使用2'-氨基-2',3'-二脱氧核苷酸-5'-磷酰咪唑化物对一系列核酸模板的复制。聚合反应在短同聚物RNA和LNA模板上迅速完成，这些模板有利于A型双链结构。我们表明，用二氨基嘌呤取代腺嘌呤碱基，用C5-(1-丙炔基)尿嘧啶取代尿嘧啶，可以产生复制效率更高的序列。最后，我们探索了更长的、混合序列RNA模板的复制，以评估2'-氨基-2',3'-二脱氧核苷-5'-磷酰咪唑化物的序列通用复制能力。我们的结果是朝着实现与原始细胞模板复制兼容的自我复制遗传聚合物迈出的重要一步，并表明N2'→P5'-磷酰胺DNA可能具有作为自我复制系统发挥作用的潜力。
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