Viral RNA, like the human transcriptome, is punctuated by infrequent but critical base modifications and non- Watson-Crick motifs. Many knowledge gaps exist in understanding where, when and why certain modifications such as pseudouridine (?) and N6-methyl-adenosine (m6A) are enzymatically written onto mRNA. Similarly, guanosine-rich regions of viral RNA and the human transcriptome that may potentially fold to G-quadruplex motifs are conserved in regulatory regions controlling translation and viral replication for reasons that remain unclear. This research project hypothesizes that secondary structural motifs such as stem-loop structures and G-quadruplexes constitute the recognition sites for RNA modification. Additionally, these sites are hotspots for oxidative modification (8-oxo-7,8-dihydroguanosine, rOG) such as occurs during oxidative stress generated by viral infections. Thus, the project will examine the interplay of base modification (pseudouridinylation, guanosine oxidation and adenosine methylation) with secondary structural motifs in RNA. New innovative chemical biology tools will be developed to sequence long mRNA strands for folded structures by examining the ability of protein nanopores to thread and translocate folded or unfolded RNA. Similarly, base modifications will be identified using specific chemistries to amplify signals from base modification.
The specific aims are to (1) investigate the sequence vs. structural motif of pseudouridine locations in ZIKV RNA, (2) sequence for rOG and correlate sites with secondary structure vs. solvent exposure, and (3) correlate G4 folds of ZIKV RNA with m6A. The human health relevance of this research is to provide foundational science for understanding the molecular choreography of mRNA, both human and viral, in order to advance health strategies combatting viral infection, cancer, and age-related disorders.
The proposed research is relevant to public health because it will provide methods for sequencing viral genomes and human transcriptomes for RNA modifications, some of which may be induced by oxidative stress or viral infection; these modifications will then be correlated to non-canonical structural motifs in nucleic acids. Oxidative stress is a causative agent in age- related disorders including cancer, diabetes, and Alzheimer?s disease, as well as playing a role in injury and inflammation-related disorders such as traumatic brain injury (TBI), and methylation markers in RNA appear to modulate viral genome replication and expression. The project is therefore relevant to NIH?s mission to understand RNA chemistry and its underlying roles in disease.
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