Template-Directed Nonenzymatic Primer Extension Using 2‑Methylimidazole-Activated Morpholino Derivatives of Guanosine and Cytidine

Efforts to develop self-replicating nucleic acids have led to insights into the origin of life and have also suggested potential pathways to the design of artificial life forms based on non-natural nucleic acids. The template-directed nonenzymatic polymerization of activated ribonucleotide monomers is generally slow because of the relatively weak nucleophilicity of the primer 3′-hydroxyl. To circumvent this problem, several nucleic acids based on amino-sugar nucleotides have been studied, and as expected, the more-nucleophilic amine generally results in faster primer extension. Extending this logic, we have chosen to study morpholino nucleic acid (MoNA), because the secondary amine of the morpholino-nucleotides is expected to be highly nucleophilic. We describe the synthesis of 2-methylimidazole-activated MoNA monomers from their corresponding ribonucleoside 5′-monophosphates and the synthesis of an RNA primer with a terminal MoNA nucleotide. We show that the activated G and C MoNA monomers enable rapid and efficient extension of the morpholino-terminated primer on homopolymeric and mixed-sequenced RNA templates. Our results show that MoNA is a non-natural informational polymer that is worthy of further study as a candidate self-replicating material.

开发自我复制核酸（self-replicating nucleic acids）的相关研究，不仅为生命起源问题提供了新的认知，也为基于非天然核酸（non-natural nucleic acids）设计人工生命形式指明了潜在路径。活化核糖核苷酸单体（activated ribonucleotide monomers）的模板指导非酶聚合（template-directed nonenzymatic polymerization）通常反应速率较慢，这是由于引物3'-羟基（primer 3′-hydroxyl）的亲核性相对较弱。为解决这一问题，学界已对多种基于氨基糖核苷酸（amino-sugar nucleotides）的核酸展开研究，且正如预期，亲核性更强的氨基通常可实现更快的引物延伸（primer extension）。基于这一思路，我们选择研究吗啉代核酸（morpholino nucleic acid, MoNA），因为吗啉代核苷酸（morpholino-nucleotides）所含的仲胺被认为具有极强的亲核性。本研究阐述了以对应核糖核苷5'-一磷酸（ribonucleoside 5′-monophosphates）为原料合成2-甲基咪唑活化吗啉代核酸单体的方法，以及制备带有末端吗啉代核苷酸的RNA引物的过程。实验结果表明，活化的G型与C型吗啉代核酸单体可在均聚及混合序列RNA模板上，实现末端带有吗啉代基团的引物的快速高效延伸。本研究结果证实，MoNA是一种非天然信息聚合物，有望作为候选自我复制材料开展进一步研究。