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

凭借我们的第一笔发现基金，我们开发了一种等温DNA扩增方法，该方法由复制链中不稳定基团的存在驱动。在我们的节目中，我们将广泛探讨这个简单的原理的普遍性，放大的不稳定。在项目一中，我们将开发不同的方法从外部触发这种损伤诱导的DNA扩增过程。引入外部触发器的好处是双重的。首先，系统化学的一个方面涉及到制造反应性的组件或网络。我们的复制系统是DNA机器的一个漂亮的例子，因为复制周期中的周转源于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做出反应，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，通过屏蔽不稳定群，我们可以最小化LIDA的背景源，即在没有任何目标序列的情况下复制子链的伪钝端结扎。（要复制的序列，这应该是启动复制所必需的）。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，我们将以基于我们的病变诱导方法的聚合酶链反应（PCR）的初步数据为基础。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周转的自我复制系统的例子。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对系统化学和益生元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的优势，因为它们可以用来确定存在哪种感染因子菌株。由于我们的系统专注于扩增小靶标，因此在某些应用中我们将比其他PCR系统具有优势。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/或结扎。由于表面已被证明选择性地与核碱基结合，并促进磷酸键，我们很兴奋地发现病变如何影响这些过程。我们假设碱基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改善周转。同时，它可能允许不太完美的链更多地与溶液中的物种相互作用，这可能有助于它选择更完美的拷贝。与此同时，我们将使用固定化DNA和多路复用的可能性来研究LIDA的表面版本。

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.