Mechanistic studies of cation catalysis in nonenzymatic RNA primer extension
Citation
Pazienza, Lydia Trinidad. 2022. Mechanistic studies of cation catalysis in nonenzymatic RNA primer extension. Doctoral dissertation, Harvard University Graduate School of Arts and Sciences.Abstract
The origin of life is believed to have centered around RNA acting as both geneticinformation and catalyst. Several decades into research around the RNA world hypothesis,
however, life has yet to be created synthetically. This is in part due to the difficulty of
transitioning from the prebiotic synthesis of RNA monomers to RNA oligonucleotides, and the
subsequent replication of RNA. Only once RNA can undergo replication nonenzymatically and
react to the lengths of ribozymes can the full potential of this theory be evaluated. While much
has been learned mechanistically about nonenzymatic RNA copying in the last several years, a
particular gap in knowledge surrounds the role of metal ion catalysis in nonenzymatic RNA
primer extension, due to the weak and transient nature of this catalytic interaction.
This dissertation aims to characterize the nature of the interactions of cations in primer
extension. In Chapter 1, the history of prebiotic chemistry is reviewed, as is what is known about
RNA-cation interactions. In Chapter 2, I describe the metal ion, kinetics, and binding studies that
support the model of 3′-OH nucleophilic activation from an inner-sphere contact with a catalytic
cation. I additionally show the sequence, structural, and cation dependent effects of metal ion
interactions in primer extension. I further support the previously published evidence shown
indicating the 3′-OH is deprotonated before the rate-limiting step through kinetic isotope effects,
and exclude the role of a metal-bound hydroxide species. Lastly, I measure the relative binding
of metal ions as affected by the distance of the reacting bridged dinucleotide, suggesting an
interaction with an oxygen of the bridged dinucleotide and the catalytic metal ion. In Chapter 3, I
further validate this metal-bridged dinucleotide interaction as being relevant for primer extension
through thiophosphoroimidazolide studies and metal rescue. We show that only one product is observed in the presence of Mg2+ in crystallo, regardless of starting material identity. Due to
racemization of the starting material over the time scale of crystallography, this observation
supports that only one diastereomer is reactive for primer extension, supporting a metal ion
interaction with one specific prochiral oxygen on phosphorous, and an SN2-like mechanism for
primer extension. Through this mechanistic understanding, the stereochemistry of the
diastereomers can be assigned, and the coordination geometry of the catalytic metal ion is
proposed, allowing chelation development to be performed intelligently. In Chapter 4, we
address the possibility of Mn2+ as a prebiotically plausible catalyst in the RNA world, and find it
improves ligation, primer extension rates (homo-polymeric and mixed template), and reaction
yields, despite an increase in hydrolysis of reacting bridged dinucleotide. However, the increase
in reactivity in the presence of Mn2+ results in a loss in fidelity at comparable concentrations of
metal ion used for Mg2+, likely due to increased competition of activated monomers (which react
with lower fidelity), supporting pre-existing hypotheses on fidelity for nonenzymatic primer
extension.
In sum, the results in this dissertation show the importance of metal ions in nonenzymatic
primer extension, provide experimental results to support a model of cation catalysis, and
provide a scaffold for designing co-catalysts to better enable metal ion-RNA interactions to
improve nonenzymatic primer extension.
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