Synthetic Biology Methods to Optimise T4 RNA Ligase Activities

Easey, Amy (2018) Synthetic Biology Methods to Optimise T4 RNA Ligase Activities. Doctoral thesis, University of East Anglia.

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The T4 nucleic acid (NA) ligases repair breaks in nucleic acids, which can be double-stranded DNA, RNA or a combination of both. The bacteriophage T4 has 3 NA ligases, one is a DNA ligase (T4Dnl) and two are RNA ligases: T4 RNA ligase 1 (T4Rnl1) and T4 RNA ligase 2 (T4Rnl2). These T4 DNA and RNA ligases are widely used as tools in a number of molecular biology techniques. A structural bias has been reported when the T4 RNA ligases are used in next generation sequencing (NGS) adapter ligation and there are issues with nucleic acid manipulation; these biochemical activities could be improved. Synthetic biology techniques were used to engineer a number of novel enzymes by combining the ligases with other proteins which have specific biochemical activities; and by mutating amino acid residues. A total of 22 novel enzymes have been constructed and tested to check changes in ligation activity and additional functions of the ligase fusions. This includes 8 ligases with specific amino acid changes to motif I, 4 ligases with nucleotide binding domain swaps, 3 adenylate kinase (AK)-T4 NA ligase fusions, 3 polynucleotide kinase (Pnk)-T4 NA ligase fusions and 3 fusions with truncated Mycobacterium smegmatis LigD (MSLigD) and the T4 NA ligases.
Ligation analysis revealed that a number of the fusion ligases were functional, and exhibited novel biochemical activities. This included utilising a new co-factor for the AK-ligase fusions, where ADP was used instead of ATP for ligation. Lower quantities of AKRnl1 were required to achieve the same ligation as T4Rnl1, an interesting feature for industrial retail. The Pnk-ligase fusions correctly ligated substrates that could not be ligated by the original T4 NA ligases, where the DNA/RNA substrate break had 3’ phosphate instead of 5’ phosphate. The amino acid mutations indicated the published sequences for the conserved polypeptide motifs are often lacking important surrounding amino acids. Fusions of T4Dnl with truncated MSLigD offered a way to use higher concentrations of ATP for ligation, in high ATP concentrations MsLigD dissociates from the DNA and is unable to complete the ligation reaction. Optimisation studies need to be completed for the new functional ligase fusions and the modified T4 NA ligases, considering temperature, buffer constituents and reaction time. The ligases detailed in this body of work offer exciting examples of creating fusion multi-function enzymes with novel activities. These new ligases may become alternative tools for molecular techniques, especially in NGS adapter ligation. This PhD work is part of a BBSRC iCASE studentship with Inspiralis (Norwich Research Park Innovation Centre, Colney Lane, Norwich NR4 7GJ) who retail T4 nucleic acid ligases.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Stacey Armes
Date Deposited: 12 Apr 2019 12:09
Last Modified: 12 Apr 2019 12:09


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