Methods of partitioning, biogenesis, and selecting for natural and engineered CoA-RNA
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More than a decade ago, RNAs with NAD+, CoA, and acylated CoA caps were identified. Since then, studies have described NAD+'s protective, cap-like function in bacteria and its role in promoting mRNA degradation in eukaryotic cells. However, the identities, functional roles, and mechanisms of biogenesis of CoA-RNA have not yet been explored. NAD-RNAs are generated primarily by co-transcriptional capping where NAD+ is inserted into the +1 position of transcripts in place of ATP. However, this co-transcriptional model is unlikely to generate CoA-RNAs in cells because the required non-canonical initiator nucleotide for co-transcription, 3' dephospho CoA (dpCoA), is estimated to be ~200 fold less concentrated in cells than NAD+ and is therefore unlikely to outcompete ATP for the +1 position of transcripts. Thus, this work demonstrates that post-transcriptional capping by enzyme phosphopantetheine adenylyltransferase (PPAT) is a possible mechanism to generate CoA-RNA. Additionally, because having a reliable method to partition CoA-RNAs from other total RNA is a crucial step for studying and making use of them, this work describes the development of a CoA Capture Seq method for separating CoA-RNAs from total RNA. Although the CoA Capture Seq method described in this work needs further optimization before it is suitable for identifying endogenous CoA-RNAs, it was adapted and used successfully to establish in vivo post-transcriptional capping of RNAs by PPAT to generate CoA-RNAs. I further investigated methods of in vivo biogenesis of CoA-RNA by performing a selection under in vivo like conditions to select for RNAs capable of capping themselves with 4' phosphopantetheine (pPant) to become CoA-RNAs (CoAzymes) and RNAs which serve as the best substrate to be capped enzymatically by PPAT. The selection conditions were designed to mimic intracellular conditions (neutral pH, fewer number of ions included, lower ion concentrations, etc) to increase the probability of selecting for RNAs which retained functionality in cells. Before starting the selection, five different reverse-transcriptases (RTs) were tested and optimized under various reaction conditions with RNA library templates of varying structure, to determine which RT introduced the least amount of inter-library bias. The RT analysis revealed that BST 3.0 DNA Polymerase was the best choice for the RT step of the selection due to its excellent processivity, significant yield, and low-inter library bias. After 12 rounds of selection, no significant increase in CoAzyme or PPAT capping activity was observed, thus selection rounds were prepared for HTS to evaluate the library pool's progression throughout the course of the selection. Unfortunately, the HTS analysis revealed no convergence or enrichment of specific sequences or clusters of sequences. Additionally the diversity of sequence reads in each round was also inconsistent and the enrichment analysis revealed the inconsistencies in population structure throughout the selection. These data suggest the selection failed, which is likely related to the overly stringent selection parameters, especially the buffer conditions. Overall, this work illustrates the importance of selection parameters, especially the selection buffer and using RTs that introduce minimal bias, for successful selection outcomes.
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Ph. D.