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

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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.