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

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。随着对整个基因组进行测序的能力变得更快、更便宜，这场革命仍在继续，这无疑为更个性化的医学铺平了道路。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先者已经确定了构建核苷酸构建块和激活核苷酸以进行模板聚合的可行策略。然而，仍然存在的一个问题是，复制后产生的产物DNA双链本质上非常稳定。例如，自然热循环以及一些其他机制被用来调节DNA产物双链稳定性，从而导致复制过程中的周转。我们实验室已经开发出唯一的等温连接链式反应(病变诱导DNA扩增-LIDA)，该反应表现出快速、指数复制动力学。我们在理解LIDA动力学方面的最新进展揭示了该酶的不同作用：它不仅加速了连接反应，而且比产物双链更稳定中间双链。这一发现是实现产生非酶自复制寡核苷酸系统这一突出目标的关键。在这个建议中，我们描述了我们的计划，即制造一种插层催化剂，选择性地稳定产物双链上的相同中间体。我们将使用在速度上与T4 DNA连接酶相当的化学连接方法来产生一个非酶复制系统。我们还将探索其他可能存在于益生菌地球上的外部调节，如控制离子单价和二价氧化还原状态存在的氧化还原梯度，每种状态对DNA杂交具有非常不同的稳定作用。我们还将探索矿物对LIDA反应中竞争的影响，以及通过连接较小的复制子来使用LIDA进行DNA聚合。最后，我们将探索将LIDA用于另一个研究领域：基于DNA材料的纳米组件的自我复制。作为唯一已知的等温连接链式反应，我们的系统完全准备提供一种复制基于DNA的形状、笼子和管的机制，就像那些由麦吉尔大学的Sleiman实验室开创的那样。创造基于DNA的自我复制的纳米材料将标志着仿生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(SNAs)选择屏幕相结合的能力，以探索基于比大多数适配子小得多的单个结合序列来选择多价结合剂的能力。我们假设，我们可以基于SNAs上高密度的DNA促进的多价相互作用实现高亲和力。

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.