Stepwise Reconstitution of RNA Cellular Activity

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2022

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Modern biomedical science enjoys an unprecedented ability to identify and describe viral pathogenic mechanisms, as well as characterize the biomolecular components that constitute them. However, we have not yet achieved a fully quantitative biophysical understanding in which modeling of component molecules is accurately predictive of viral functions in cells. Reconstitution, in which a cellular process is reduced to the in vitro behavior of its component parts, is a promising strategy for establishing such predictive relationships, but there is debate as to whether the structures and activities of RNAs observed in vitro transfer to the cellular context. My thesis work seeks to probe these predictive relationships between in vitro and in vivo activity of viral RNAs and apply them to viral RNA-targeted drug development. I first quantitatively examined the relationships among levels of reconstitution of the human immunodeficiency virus 1 (HIV-1) cellular process of transactivation. This process involves complex RNA-protein interactions between an HIV-1 RNA, the transactivation response element (TAR), and a complex of the viral Tat protein and host super-elongation complex proteins (SEC). I designed library of seventeen TAR mutants and quantitatively measured their activity using four different assays, including a gene-reporter cell-based assay, electrophoretic mobility shift assay, fluorescence-based binding assay, and interhelical stacking measurements using nuclear magnetic resonance imaging (NMR) to probe behavior at varying levels of reconstitution. I found strong agreement (r=0.96-0.86) when comparing each level such that cellular activity was predicted by the reconstituted protein complex binding in vitro, which was predicted by peptide binding, which was then predicted by the stacking dynamics of the unbound RNA. This work demonstrates that reconstitution of complex cellular processes at multiple levels of reduction is a viable strategy for establishing predictive relationships with high fidelity. After establishing this predictive relationship of HIV-1 TAR ensemble behavior in vitro and in cells, I looked to take advantage of this well studied system for drug-screening applications. In reviewing the history of small molecule (SM) TAR-binders and other viral helix-junction-helix (HJH) motifs, it struck me that very few of the many compounds that bound TAR with high affinity in vitro had significant if any activity in cells. This contrasts with what would be expected based on my previous study, which demonstrates a highly predictive relationship between in vitro and cellular activities of TAR. This must mean that the discrepancy in activities must not be due to TAR, but instead due to the small molecules themselves. I investigated this idea further by executing a study of small molecules that have been reported to bind to various helix-junction-helix motif RNAs, other than TAR, and measuring their activity in vitro and in cells against both TAR and another distinct HIV-1 HJH RNA, RRE. I found that all of the molecules bound both TAR and RRE, some with equivalent affinities to their intended target. To further investigate the structural basis of this nonspecific RNA-binding behavior, I performed a structural survey of all RNA-SM complexes in the Protein Data Bank (PDB). This revealed that RNA-SM interactions with known high specificity differed in hydrogen bonding (H-bond) patterns from known low specific interactions. High specificity interactions all had unique H-bond patterns, which low specific interactions were marked by a similar H-bond pattern to atoms that are exposed in canonical A-form RNA helices. The result of this study suggests that HIV-1 TAR in its ground state form is not necessarily a good candidate for drug targeting, as the SM that are able to bind to it will likely bind to many other cellular RNAs, making in vitro screening a poor predictor of cellular activity. With this knowledge, I instead focused my drug-targeting efforts on a structurally complex RNA with unique targetable motifs – the Zika virus (ZIKV) Xrn1-exonuclease resistant RNA element (xrRNA1). Using a pipeline consisting of structure-based virtual screening and experimental screening of viral attenuation and cellular function, I screened ~80,000 small molecules and found eight that significantly modified viral replication in cells, with modest effects on cellular activity of xrRNA1. I have also included a separate project I worked on during my PhD, development of a curriculum around cultural determinants of health and health disparities in the School of Medicine.

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Kelly, Megan Leigh (2022). Stepwise Reconstitution of RNA Cellular Activity. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/25311.

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