Evolution and Evaluation of Engineered DNA Ligases for Improved Blunt-End Ligation

<p>DNA ligases are fundamental enzymes in molecular biology and biotechnology where they perform essential reactions, e.g. to create recombinant DNA and for adaptor attachment in next-generation sequencing. T4 DNA ligase is the most widely used commercial ligase owing to its ability to catalyse ligation of blunt-ended DNA termini. However, even for T4 DNA ligase, blunt-end ligation is an inefficient activity compared to cohesive-end ligation, or its evolved activity of sealing single-strand nicks in double-stranded DNA. Previous research from Dr Wayne Patrick showed that fusion of T4 DNA ligase to a DNA-binding domain increases the enzyme’s affinity for DNA substrates, resulting in improved ligation efficiency. It was further shown that changes to the linker region between the ligase and DNA-binding domain resulted in altered ligation activity. To assist in optimising this relationship, we designed a competitive ligase selection protocol to enrich for engineered ligase variants with greater blunt-end ligation activity. This selection involves expressing a DNA ligase from its plasmid construct, and ligating a linear form of its plasmid, sealing a double-strand DNA break in the chloramphenicol resistance gene, permitting bacterial growth. Previous researcher Dr Katherine Robins created two linker libraries of 33 and 37 variants, from lead candidate ligase-cTF and (the less active form of p50-ligase variant) ligase-p50, respectively. Five rounds of selection were applied to each library. One linker variant, denoted ligase-CA3 showed the greatest improvement, comprising 42% of the final selected ligase-cTF population. In contrast, a lead linker variant from the ligase-p50 library was not obtained. In this study one additional round of selection was applied to the ligase-p50 library to test whether a lead variant would emerge. However, the linker variants selected at the end of Round 6 did not suggest a clear lead candidate, so one of the top variants (ligase-PPA17) was selected to represent this population in a fluorescence-based ligation assay that I optimised. Following identification of optimal reaction buffers to improve protein stability and DNA ligation, six engineered variants were compared for blunt-, cohesive-end, and nick sealing ligation activities. All five engineered variants exhibited improved blunt-end ligation activity over T4 DNA ligase. Ligase-PPA17 (1.9-fold improvement over T4 DNA ligase) was best performing for blunt-end ligation. This study found no evidence that ligase-CA3 was significantly improved over its predecessor, ligase-cTF in blunt-end ligation, however it was the best performing variant at cohesive-end ligation. Overall, we have evolved DNA ligase variants with improved blunt-end ligation activity over T4 DNA ligase which may be more advantageous in molecular biology and biotechnology for a variety of applications.</p>

Evolution and Evaluation of Engineered DNA Ligases for Improved Blunt-End Ligation

<p>DNA ligases are fundamental enzymes in molecular biology and biotechnology where they perform essential reactions, e.g. to create recombinant DNA and for adaptor attachment in next-generation sequencing. T4 DNA ligase is the most widely used commercial ligase owing to its ability to catalyse ligation of blunt-ended DNA termini. However, even for T4 DNA ligase, blunt-end ligation is an inefficient activity compared to cohesive-end ligation, or its evolved activity of sealing single-strand nicks in double-stranded DNA. Previous research from Dr Wayne Patrick showed that fusion of T4 DNA ligase to a DNA-binding domain increases the enzyme’s affinity for DNA substrates, resulting in improved ligation efficiency. It was further shown that changes to the linker region between the ligase and DNA-binding domain resulted in altered ligation activity. To assist in optimising this relationship, we designed a competitive ligase selection protocol to enrich for engineered ligase variants with greater blunt-end ligation activity. This selection involves expressing a DNA ligase from its plasmid construct, and ligating a linear form of its plasmid, sealing a double-strand DNA break in the chloramphenicol resistance gene, permitting bacterial growth. Previous researcher Dr Katherine Robins created two linker libraries of 33 and 37 variants, from lead candidate ligase-cTF and (the less active form of p50-ligase variant) ligase-p50, respectively. Five rounds of selection were applied to each library. One linker variant, denoted ligase-CA3 showed the greatest improvement, comprising 42% of the final selected ligase-cTF population. In contrast, a lead linker variant from the ligase-p50 library was not obtained. In this study one additional round of selection was applied to the ligase-p50 library to test whether a lead variant would emerge. However, the linker variants selected at the end of Round 6 did not suggest a clear lead candidate, so one of the top variants (ligase-PPA17) was selected to represent this population in a fluorescence-based ligation assay that I optimised. Following identification of optimal reaction buffers to improve protein stability and DNA ligation, six engineered variants were compared for blunt-, cohesive-end, and nick sealing ligation activities. All five engineered variants exhibited improved blunt-end ligation activity over T4 DNA ligase. Ligase-PPA17 (1.9-fold improvement over T4 DNA ligase) was best performing for blunt-end ligation. This study found no evidence that ligase-CA3 was significantly improved over its predecessor, ligase-cTF in blunt-end ligation, however it was the best performing variant at cohesive-end ligation. Overall, we have evolved DNA ligase variants with improved blunt-end ligation activity over T4 DNA ligase which may be more advantageous in molecular biology and biotechnology for a variety of applications.</p>

Ligation and supercoiling of plasmids containing single strand interruptions

We have developed a simple procedure for determining if a plasmid DNA contains single strand interruptions that can be sealed by DNA ligase and thus contain single strand interruptions with 5'-phosphate and 3'-hydroxyl groups. In this procedure, plasmid DNAs with ligatable nicks were ligated by T4 DNA ligase, supercoiled by E. coli DNA gyrase and analyzed by agarose gel electrophoresis. Our results show that after sealing nicked circular DNA with DNA ligase, supercoiling by DNA gyrase produces a rapidly migrating DNA species that can easily be distinguished from nicked circular DNA by agarose gel electrophoresis.

CLONING OF SHRNA SEQUENCE SPECIFIC TO ELASTASE OF MACROPHAGES INTO EXPRESSION VECTOR PGPV-17019250

BACKGROUND: Macrophage elastase/MMP12 is one of the most important matrix metalloproteinase that cells secrete into the extracellular space. Secreted and activated MMP12 is capable to digest the proteins of extracellular matrix, such as fibronectin, laminin and elastin, facilitating the migration of immune cells to the inflamed tissue. AIM: The aim of our study was to create a vector that would express a specific small interfering RNA (shRNA) to suppress the expression of human macrophage elastase. METHODS: The online tool "siRNA Wizard" was used to design cDNA encoding shRNA specific to human macrophage elastase. The restriction endonucleases BamH1 and EcoRI, as well as a commercial T4 DNA ligase we used to clone the named cDNA into the expression vector pGPV-17019250. The presence of the cDNA in the vector was confirmed by PCR and DNA-sequencing with vector-specific primers. RESULTS: In this study, we obtained the sequence of cDNA, encoding shRNA specific to macrophage elastase. We also cloned the named cDNA into the expression vector pGPV-17019250 and confirmed its exact location in the vector. CONCLUSION: The generated expression vector pGPV-17019250-EM designated to suppress human macrophage elastase in cultured cells.

Structural Landscape of the Transition from an ssDNA Dumbbell Plus Its Complementary Hairpin to a dsDNA Microcircle Via a Kissing Loop Intermediate

The experimental construction of a double-stranded DNA microcircle of only 42 base pairs entailed a great deal of ingenuity and hard work. However, figuring out the three-dimensional structures of intermediates and the final product can be particularly baffling. Using a combination of model building and unrestrained molecular dynamics simulations in explicit solvent we have characterized the different DNA structures involved along the process. Our 3D models of the single-stranded DNA molecules provide atomic insight into the recognition event that must take place for the DNA bases in the cohesive tail of the hairpin to pair with their complementary bases in the single-stranded loops of the dumbbell. We propose that a kissing loop involving six base pairs makes up the core of the nascent dsDNA microcircle. We also suggest a feasible pathway for the hybridization of the remaining complementary bases and characterize the final covalently closed dsDNA microcircle as possessing two well-defined U-turns. Additional models of the pre-ligation complex of T4 DNA ligase with the DNA dumbbell and the post-ligation pre-release complex involving the same enzyme and the covalently closed DNA microcircle are shown to be compatible with enzyme recognition and gap ligation.

Design and Synthesis of Ag Nanocluster Molecular Beacon for Adenosine Triphosphate Detection

This study presents a fluorescence method for detecting adenosine triphosphate (ATP) based on a label-free Ag nanocluster molecular beacon (MB) with high sensitivity. The sensor contains a hairpin-shaped MB, two short single-stranded DNA strands, and T4 DNA ligase. The MB consists of three parts, which are the template DNA sequence for synthesizing Ag nanoclusters at the 5′ end, the middle DNA with a hairpin-shaped structure, and the guanine base-rich DNA sequence at the 3′ end. The sensor exhibits high fluorescence intensity in the absence of ATP. However, when the probe is used for ATP detection, the two short DNA sequences in the sensor would form a long sequence by enzymatic ligation reaction; this long sequence opens the hairpin-shaped structure of the MB and decreases the fluorescence of the system. Under optimal analytical conditions, a clear linear relationship is observed between ATP concentration and fluorescence intensity in the range of 0.1–10 μM. The interference presented by other small molecules during ATP detection is evaluated, and results confirm the good selectivity of the proposed sensor. Compared with traditional methods, the sensor is label free, easy to operate, inexpensive, and highly sensitive.