Hicks, JoannaKamal, Aliyah2026-01-292026-01-292025-09-14https://hdl.handle.net/10289/17905MicroRNAs (miRNAs) are short regulatory RNAs that play critical roles in post-transcriptional gene regulation. Dysregulation of miRNA expression can be implicated in numerous diseases, including cancer, making miRNAs valuable diagnostic and prognostic biomarkers. Next-generation sequencing (NGS) enables quantitative profiling of miRNA populations, but a key limitation lies in the adapter ligation step required for library preparation. Current commercial RNA ligases, typically active between 14-37 ℃, display strong biases based on RNA sequence and secondary structure resulting in a skewed representation of miRNA populations. At low temperatures, miRNA secondary structures can obscure 5’-phosphate, and 3’-hydroxyl ends, preventing efficient ligation. Thermophilic RNA ligases with high thermostability and distinct substrate preferences may therefore reduce ligation bias and improve NGS accuracy. The archaeal Paleococcus pacificus RNA ligase (PpaRnl) represents a valuable model for exploring the structural and mechanistic features of thermophilic RNA ligases. This study aimed to further investigate the substrate preference of a PpaRnl mutant; K238G, a point mutation of lysine at position 238, a key residue in the coordination of the AMP and in turn the binding of the Mg2+ ions in PpaRnl. Alongside the structural and mechanistic characterisation of several PpaRnl mutants to explore the role of a newly identified Mg2+ binding site (Mg2+ B) close to the active site. Key residues for coordinating the Mg2+B ion were mutated in the PpaRnl enzyme (D94A, D248A, E155A, and the double mutant D94A-D248A) as characterisation of these mutants would allow for a deeper understanding of this mechanism. Mutants were expressed in Escherichia coli and purified using a combination of immobilised metal affinity chromatography (IMAC) and gel-filtration chromatography. Expression and purification outcomes varied significantly between constructs, while some mutants were successfully obtained in sufficient yields, others demonstrated poor or inconsistent expression, often not even seeing improvement after optimisation of growth conditions and large-scale culture volumes. In particular, the E155A mutant displayed extremely slow growth, while the D94A-D248A double mutant produced negligible results across expression trials. Further characterisation of the mutants, K238G, D94A, and D248A, had shown that these enzymes retain robust activity between 60-65 ℃, with measurable activity extending up to 80 ℃. Distinct sequence and cofactor preferences were observed. When combined with adapters, these mutants efficiently produced RNA ligation products with minimal side reactions and, in several cases, displayed improved activity compared to the wild-type enzyme. The structures of PpaRnl D94A and D248A were solved via X-ray crystallography and although there was similarity in the structures compared to that of the wild-type enzyme and K238G, it was found that a number of the residues coordinating the ligands present within the active-site had changed their orientation. This resulted in the Mg2+B ion not being present in D94A, and no AMP cofactor, Mg2+A or Mg2+B present within the D248A mutant. Overall, this work highlighted the variability in expression and crystallisation outcomes among the second Mg2+ binding site mutants of PpaRnl with notable differences observed in expression, activity, and structure, highlighting that the Mg2+B ion and its coordination have an impact of the efficiency of the enzyme. This unique mechanism is still not fully understood but its importance and impact has been highlighted within this research, reiterating the importance of construct specific optimisation in structural studies and the potential utility of engineered thermophilic RNA ligases, as tools to reduce ligation bias in NGS based miRNA sequencing. Allowing for the possible discovery of biomarkers within viral diseases, and cancer.enAll items in Research Commons are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.RNA LigaseThermophilic enzymestructural biologybiochemistryThesis