DNA Amplification Beyond Biology: Achieving Self-Replication in Nano-Assemblies and Prebiotic Model Systems

超越生物学的 DNA 扩增：在纳米组件和益生元模型系统中实现自我复制

• 批准号: RGPIN-2020-05976
• 负责人: Gibbs, Julianne
• 金额: $ 3.5万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=718136
• 关键词: DNA; Amplification; Beyond; Biology; Achieving

The ability to control DNA amplification processes through the judicious use of enzymes and nucleotide building blocks has revolutionized biodiagnostics. This revolution continues as the ability to sequence entire genomes becomes faster and cheaper, undoubtedly paving the way to more personalized medicine. How DNA and RNA replication arose, however, remains a major subject of research. Current leaders in origins of life research have identified plausible strategies for building nucleotide building blocks and activating nucleotides for templated polymerization. One problem that still remains, however, is that the product DNA duplex that results after replication is intrinsically very stable. As such natural thermal cycling, as well as some other mechanisms, have been invoked to modulate DNA product duplex stability such that turnover in the replication process can ensue. Our lab has developed the only isothermal ligase chain reaction (Lesion-Induced DNA Amplification-LIDA) that exhibits rapid, exponential replication kinetics. Our recent progress at understanding the kinetics of LIDA has uncovered the varied role of the enzyme: not only does it accelerate the ligation reaction but it also stabilizes the intermediate duplex more so than the product one. This discovery is key to achieving an outstanding goal of generating a non-enzymatic self-replication oligonucleotide system. In this proposal we describe our plans to make an intercalator catalyst that selectively stabilizes the same intermediate over the product duplex, which we will use with chemical ligation methods comparable in rate to that of T4 DNA ligase to generate a nonenzymatic replicating system. We will also explore other possible external modulations that could have been present on prebiotic earth like redox gradients that control the presence of monovalent and divalent redox states of ions, each with very different stabilizing effects on DNA hybridization. We will also explore the impact of minerals on competition in LIDA reactions, as well as the use of LIDA to perform DNA polymerization by ligation of smaller replicators. Finally, we will explore using LIDA in another realm of reseach: self-replication of nanoassemblies based on DNA materials. As the only known isothermal ligase chain reaction, our system is perfectly poised to provide a mechanism for replicating DNA-based shapes, cages and tubes, like those pioneered by the Sleiman lab at McGill University. Creating self-replicating DNA-based nanomaterials will mark a major milestone in the development of life-like synthetic systems. We will also explore the ability to couple DNA amplification with selection screens for spherical nucleic acids (SNAs) to explore the selection of multivalent binding agents based on individual binding sequences much smaller than most aptamers. We hypothesis that we can achieve high affinity based on the multivalent interactions facilitated by the high density of DNA on the SNAs.

通过明智地使用酶和核苷酸构建块来控制DNA扩增过程的能力已经彻底改变了生物诊断学。这场革命还在继续，因为对整个基因组进行测序的能力变得更快、更便宜，无疑为更个性化的医疗铺平了道路。然而，DNA和RNA复制是如何产生的仍然是一个主要的研究课题。目前生命起源研究的领导者已经确定了构建核苷酸构建模块和激活核苷酸进行模板聚合的合理策略。 然而，仍然存在的一个问题是，复制后产生的产物DNA双链体本质上非常稳定。 因此，已经调用天然热循环以及一些其他机制来调节DNA产物双链体稳定性，使得复制过程中的周转可以随之发生。我们的实验室已经开发出唯一的等温连接酶链反应（病变诱导的DNA扩增-LIDA），表现出快速，指数复制动力学。我们最近在理解利达动力学方面的进展揭示了这种酶的不同作用：它不仅加速连接反应，而且比产物更稳定中间双链体。 这一发现是实现产生非酶促自我复制寡核苷酸系统的突出目标的关键。 在该提案中，我们描述了我们的计划，使嵌入催化剂，选择性地稳定相同的中间体的产品双链体，我们将使用化学连接方法的速率可比的T4 DNA连接酶，以产生一个非酶复制系统。 我们还将探索其他可能的外部调制，可能已经存在于前生物地球上，如氧化还原梯度，控制离子的一价和二价氧化还原状态的存在，每一种都对DNA杂交有非常不同的稳定作用。我们还将探讨矿物质对利达反应中竞争的影响，以及使用利达通过连接较小的复制子来进行DNA聚合。 最后，我们将探讨使用利达在另一个领域的研究：自我复制的纳米组装的基础上的DNA材料。 作为唯一已知的等温连接酶链式反应，我们的系统完全准备好提供一种复制基于DNA的形状，笼和管的机制，就像麦吉尔大学Sleiman实验室开创的那样。 创造基于DNA的自我复制纳米材料将标志着类生命合成系统发展的一个重要里程碑。 我们还将探索将DNA扩增与球形核酸（SNA）的选择筛选相结合的能力，以探索基于比大多数适体小得多的单个结合序列的多价结合剂的选择。 我们假设，我们可以实现高亲和力的基础上的多价相互作用的高密度的DNA的SNA。