Taq DNA Polymerase Mutants and 2′-Modified Sugar Recognition.
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Taq DNA Polymerase Mutants and 2′-Modified Sugar Recognition.
chemical modifications to DNA, such as modification 2 ‘, is expected to increase the utility of DNA biotechnology; However, other forms of modification of DNA is limited by their inability to effectively synthesized by a DNA polymerase enzyme. Previous efforts have identified a mutant Thermus aquaticus DNA polymerase I (Taq) enzyme that can recognize DNA 2’-modified nucleotides.
While these modified nucleotides recognize the mutant enzyme, they are unable to synthesize the full length of the modified DNA; thus, further engineering is required for this enzyme. Here, we describe a comparative biochemical studies that identify the useful properties, but previously uncharacterized, this enzyme; an enzyme, SFM19, able to recognize various 2′-modified nucleotides that is broader than that previously examined, including fluoro, Azido and amino modification.
To understand the molecular origin of these differences, we also identified certain amino acids and combinations of amino acids that contribute most to the unusual activity that were previously grown. Our data indicate that the negatively charged amino acid residues 614 and mutation of steric gate, E615, to Glycine create the optimal combination for the synthesis of modified oligonucleotides. The study provides a better understanding of the origins of the mutation of 2′-modified substrate recognition and identify SFM19 as the best candidate for advanced engineering, whether through rational design or directed evolution.
Taq DNA Polymerase Mutants and 2′-Modified Sugar Recognition.
A new iridoid, verbascoside and derivatives with inhibitory activity against Taq DNA polymerase.
DNA polymerases are enzymes that play an important role in DNA metabolism such as replication, repair, transcription, recombination, and chromosome segregation during mitosis. Here we report the isolation of a new iridoid (6-epi-catalpol, 2) and the per-O-acetyl-verbascoside (11) of the aerial parts of Buddleja cordobensis Grisebach (Buddlejaceae). Of the compound 2, we have obtained eight compounds with chemical transformations.
This group of compounds at a concentration of 500μM tested against Taq DNA polymerase. Compound 11 (per-O-acetyl-verbascoside) was the most active with IC50 of 1.21 ± 0.18μM; Compound 9, 2 and 8 are strong inhibitors with IC50 values of 5:57 ± 0.70, 21.62 ± 0.22 and 78.13 ± 0.93μM, respectively. Compound 11 and 9 could be the structure of a new leader for the development of anticancer drugs chemotherapy and useful tools to investigate the activity of DNA polymerase.
Oriented methods and activity-preserved immobilization of biologically active proteins based on the concept of active-site masking and previously reported kinetic control. We extended our research and found that hydrophobic surfaces have a noticeable effect on the activity of the active-protected site move (PIM) Taq DNA polymerase in a mixture of assembled monolayer (SAM) is produced on the Au surface. Hydrophobic SAM created using 12-mercaptododecanoic acid and 1-heptanethiol. Taq DNA polymerase activity generated is measured by PCR amplification and compared with previously reported values obtained with the hydrophilic SAM. The maximum activity of the immobilized Taq DNA polymerase was achieved in 17.5% of 12-mercaptododecanoic acid and within 90 minutes of reaction time is higher than that obtained with the hydrophilic SAM; The maximum activity by 5% 12-mercaptododecanoic acid and at 10 minutes. To apply to a commercial level, moving enzymes have to be stable, reusable and can be stored.
PIM stable Taq DNA polymerase attached to the surface and can be used for as many as 10 runs PCR comparable with enzyme-phase solution. Even after 56 days of storage at 4 ° C, the immobilized enzyme classified as 70% of the initial PIM enzyme activity. These data indicate that the PIM Taq DNA polymerase can be used for a variety of commercial applications.
