2022 Annual Meeting
Replication of Nucleic Acid Systems Mediated By a Solid-Liquid Phase Transition
The RNA world hypothesis theorizes that RNA was the precursor to both DNA and proteins, and that the earliest forms of life used only RNA for the storage of genetic material and enzymatic function. RNA molecules can only replicate using template-directed synthesis. Thus, the double-stranded RNA molecule must denature, and the single strand must act as a template to build a new molecule through ligation of monomer or oligomer nucleotides. The prebiotic challenge to this process is known as the strand inhibition problem. To denature the original double-stranded RNA molecule, the temperature is increased above the melting temperature, allowing all strands and oligomers to exist in a single-stranded formation. However, to bind the nucleotides to the template strand, the temperature needs to be lowered below the melting temperature of the nucleotides. As temperature decreases, it is both thermodynamically and kinetically favorable for the single-stranded template RNAs to rebind to their original double-stranded partner. Thus, the primary objective of this investigation was to develop a method to overcome the strand inhibition problem and achieve continuous replication across multiple cycles. To probe these methods to circumvent the strand inhibition problem, the replication of an unstructured 80 bp DNA template was studied. Five 16mer oligomers (S1-S5 and S1'-S5' on the complement strand) were used in attempts to replicate this 80 bp DNA. The S1 and S5â² are 5â²-end labeled with Cy3 and Cy5 fluorophores, respectively, to allow for quantification of replicated products in gel electrophoretograms. In preliminary experiments, successful replication was achieved, thus circumventing the strand inhibition problem, using a transition between the liquid and solid states of this 80 bp DNA. In other words, the template, substrates, and buffer can be heated at moderate temperatures until dried down to the solid state. In this solid state, both the template and oligomers are denatured, trapped in their single-stranded confirmations. The solid is resuspended in a mixture of water and ligase enzyme. Upon analysis of gel electrophoretograms, it is evident that the rehydration of the DNA results in a replicated product. Replicated product is achieved across various template concentrations, buffer concentrations, and dry-down temperatures. In addition, when the solid-liquid phase transition is repeated across five cycles, replication is achieved each time. Overcoming strand inhibition using a prebiotically plausible method such as solid-liquid phase transition is a necessary step in origin-of-life research that must be achieved to allow for replication and Darwinian evolution to occur.