DNA Amplification Beyond Biology: Achieving Self-Replication in Nano-Assemblies and Prebiotic Model Systems

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The ability to control DNA amplification processes through the judicious use of enzymes and nucleotide building blocks has revolutionized biodiagnostics. This revolution continues as the ability to sequence entire genomes becomes faster and cheaper, undoubtedly paving the way to more personalized medicine. How DNA and RNA replication arose, however, remains a major subject of research. Current leaders in origins of life research have identified plausible strategies for building nucleotide building blocks and activating nucleotides for templated polymerization.  One problem that still remains, however, is that the product DNA duplex that results after replication is intrinsically very stable.  As such natural thermal cycling, as well as some other mechanisms, have been invoked to modulate DNA product duplex stability such that turnover in the replication process can ensue. Our lab has developed the only isothermal ligase chain reaction (Lesion-Induced DNA Amplification-LIDA) that exhibits rapid, exponential replication kinetics. Our recent progress at understanding the kinetics of LIDA has uncovered the varied role of the enzyme: not only does it accelerate the ligation reaction but it also stabilizes the intermediate duplex more so than the product one.  This discovery is key to achieving an outstanding goal of generating a non-enzymatic self-replication oligonucleotide system.  In this proposal we describe our plans to make an intercalator catalyst that selectively stabilizes the same intermediate over the product duplex, which we will use with chemical ligation methods comparable in rate to that of T4 DNA ligase to generate a nonenzymatic replicating system.  We will also explore other possible external modulations that could have been present on prebiotic earth like redox gradients that control the presence of monovalent and divalent redox states of ions, each with very different stabilizing effects on DNA hybridization. We will also explore the impact of minerals on competition in LIDA reactions, as well as the use of LIDA to perform DNA polymerization by ligation of smaller replicators.  Finally, we will explore using LIDA in another realm of reseach: self-replication of nanoassemblies based on DNA materials.  As the only known isothermal ligase chain reaction, our system is perfectly poised to provide a mechanism for replicating DNA-based shapes, cages and tubes, like those pioneered by the Sleiman lab at McGill University.  Creating self-replicating DNA-based nanomaterials will mark a major milestone in the development of life-like synthetic systems.  We will also explore the ability to couple DNA amplification with selection screens for spherical nucleic acids (SNAs) to explore the selection of multivalent binding agents based on individual binding sequences much smaller than most aptamers.  We hypothesis that we can achieve high affinity based on the multivalent interactions facilitated by the high density of DNA on the SNAs.