DNA Amplification by Destabilization: A Guide to Prebiotic Replication and A Tool for Diagnostics

DNA 去稳定扩增：生命前复制指南和诊断工具

• 批准号: RGPIN-2015-06555
• 负责人: Gibbs, Julianne
• 金额: $ 4.3万
• 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: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=690859
• 关键词: DNA; Amplification; Destabilization; Guide; Prebiotic

With our first Discovery Grant, we developed an isothermal DNA amplification method driven by the presence of destabilizing groups in the replicating strands.  In our program, we will widely explore the generality of this simple principle of amplification by destabilization.  In Project One, we will develop different ways to externally trigger this process of lesion-induced DNA amplification.  The advantage of introducing an external trigger is two fold.  First, one aspect of systems chemistry involves making responsive assemblies or networks.  Our replicating system is a beautiful example of a DNA machine as the turnover in the replication cycles stems from the destabilizing groups in the DNA, not the enzyme that is used to fuse the replicators together.  By making a system responsive to an external trigger we increase the complexity of our system, which could eventually lead to its incorporation into DNA-based networks, like "DNA walkers" or DNA circuits.  Second, by masking the destabilizing group, we can minimize the source of background in LIDA, which is the pseudo-blunt end ligation of the replicator strands in the absence of any target sequence (ie., the sequence to be replicated, which should be required to instigate replication).  By minimizing the background reaction, we can make this process more feasible as a diagnostic tool.  In Project Two, we will build on preliminary data concerning the development of a polymerase chain reaction (PCR) based on our lesion-induced approach.  This project is very exciting as there are virtually no examples of self-replicating systems that exhibit turnover via a polymerization reaction rather than a ligation reaction.  Once again as the source of turnover is simple destabilizing lesions, what we learn from this system would be of keen interest in systems chemistry and the area of prebiotic replication.  Additionally, isothermal PCR methods have large advantages in disease diagnostics, as they can be used to identify which strain of infectious agent is present. As we are system is focused on amplifying small targets, we will have an advantage over other PCR systems in certain applications. Finally, in Project Three we will explore how the presence of lesions impacts DNA polymerization and/or ligation on a mineral oxide surface.  As surfaces have been shown to bind selectively to the nucleobase, and promote phosphate linkages, we are excited to find out how lesions impacts these processes.  We hypothesize that the presence of abasic groups may weaken the interactions of the oligonucleotide with the surface, which may improve turnover by freeing up the catalytic surface and the templating strand.  Simultaneously it may allow the less perfect strand to interact more with species in solution, which may aid in its selection over more perfect copies.  Concurrently with this project, we will examine a surface version of LIDA using immobilized DNA with possibilities in multiplexing.

通过我们的第一笔发现资助，我们开发了一种等温 DNA 扩增方法，该方法是由复制链中不稳定基团的存在驱动的。  在我们的计划中，我们将广泛探索这种通过不稳定来放大的简单原理的普遍性。  在项目一中，我们将开发不同的方法来外部触发损伤诱导的 DNA 扩增过程。  引入外部触发器的优点有两个。  首先，系统化学的一个方面涉及制作响应性组件或网络。  我们的复制系统是 DNA 机器的一个美丽例子，因为复制周期的更新源于 DNA 中不稳定的基团，而不是用于将复制子融合在一起的酶。  通过使系统响应外部触发，我们增加了系统的复杂性，这最终可能导致其融入基于 DNA 的网络，如“DNA 步行者”或 DNA 电路。  其次，通过掩蔽不稳定基团，我们可以最大限度地减少 LIDA 中的背景来源，LIDA 是在没有任何目标序列（即要复制的序列，这应该是启动复制所必需的）的情况下复制链的假平末端连接。  通过最大限度地减少背景反应，我们可以使这一过程作为诊断工具更加可行。  在项目二中，我们将基于有关基于损伤诱导方法开发聚合酶链式反应 (PCR) 的初步数据。  这个项目非常令人兴奋，因为几乎没有通过聚合反应而不是连接反应表现出自我复制系统的例子。  再次，由于周转的来源是简单的不稳定损伤，我们从这个系统中学到的东西将对系统化学和生命前复制领域产生浓厚的兴趣。  此外，等温 PCR 方法在疾病诊断中具有很大优势，因为它们可用于识别存在哪种传染原菌株。由于我们的系统专注于扩增小目标，因此在某些应用中我们将比其他 PCR 系统更具优势。最后，在项目三中，我们将探讨损伤的存在如何影响矿物氧化物表面上的 DNA 聚合和/或连接。  由于表面已被证明可以选择性地与核碱基结合并促进磷酸盐连接，我们很高兴了解病变如何影响这些过程。  我们假设脱碱基基团的存在可能会削弱寡核苷酸与表面的相互作用，这可能会通过释放催化表面和模板链来提高周转率。  同时，它可能允许不太完美的链与溶液中的物种更多地相互作用，这可能有助于其选择更完美的拷贝。  与该项目同时，我们将使用具有多重可能性的固定化 DNA 来检查 LIDA 的表面版本。