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: GibbsDavis, 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: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=591134
• 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.

在我们的第一个发现基金中，我们开发了一种等温DNA扩增方法，该方法由复制链中存在的不稳定基团驱动。在我们的计划中，我们将广泛探索这种简单的不稳定扩增原理的普遍性。在项目一中，我们将开发不同的方法来从外部触发这种损伤过程，诱导的DNA扩增。引入外部触发物的优点是两方面的。首先，系统化学的一个方面涉及到制造反应性组装或网络。我们的复制系统是DNA机器的一个美丽的例子，因为复制过程中的周转循环源于DNA中的不稳定基团，而不是用于将复制子融合在一起的酶。通过使系统对外部触发器做出响应，我们增加了系统的复杂性，这最终可能导致其并入基于DNA的网络，如“DNA步行者”或DNA回路。其次，通过掩蔽不稳定基团，我们可以最小化利达的背景来源，这是在不存在任何靶序列的情况下复制子链的假平端连接（即，要复制的序列，这应该是煽动复制所必需的）。通过最小化背景反应，我们可以使这个过程作为诊断工具更可行。在项目二中，我们将建立在初步数据的基础上，有关发展的聚合酶链反应（PCR）的基础上，我们的病变诱导的方法。这个项目是非常令人兴奋的，因为几乎没有例子，自我-通过聚合反应而不是连接反应表现出周转的复制系统。再一次，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在系统化学和益生元复制领域引起浓厚的兴趣。此外，等温PCR方法在疾病诊断中具有很大的优势，因为它们可用于鉴定存在哪种感染原。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具有优于其他PCR系统的优势。最后，在项目三中，我们将探索损伤的存在如何影响无机氧化物表面上的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，并促进磷酸键合，我们很高兴地发现损伤如何影响这些过程。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。与此同时，我们将使用具有多重可能性的固定化DNA来研究表面版本的利达。