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

通过我们的第一个发现基金，我们开发了一种等温DNA扩增方法，该方法由复制链中存在的不稳定基团驱动。 在我们的计划中，我们将广泛探讨这个简单的不稳定放大原理的普遍性。 在项目一中，我们将开发不同的方法来外部触发这种损伤诱导的DNA扩增过程。 引入外部触发器的优点有两个方面。 首先，系统化学的一个方面涉及制造响应组件或网络。 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电路。 其次，通过掩蔽去稳定化基团，我们可以最小化利达中的背景来源，其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，将被复制的序列，这应该是发起复制所需要的）。 通过最小化背景反应，我们可以使该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，因为几乎没有自我复制系统的例子，通过聚合反应，而不是连接反应表现出营业额。 再一次，由于营业额的来源是简单的不稳定病变，我们从这个系统中学到的东西将是系统化学和益生元复制领域的浓厚兴趣。 此外，等温PCR方法在疾病诊断中具有很大的优势，因为它们可以用于鉴定存在哪种感染原菌株。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具有优于其他PCR系统的优势。最后，在项目三中，我们将探讨病变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，并促进磷酸连接，我们很高兴地发现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营业额。 同时，它可以允许不太完美的链与溶液中的物种进行更多的相互作用，这可能有助于选择更完美的拷贝。 与该项目同时进行的是，我们将使用固定化DNA研究表面版本的利达，并具有多路复用的可能性。

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.