34 research outputs found
The clinical use of Kampo medicines (traditional Japanese herbal treatments) for controlling cancer patients’ symptoms in Japan: a national cross-sectional survey
Validation of a Multiplex Real-Time PCR Assay for Detection of Mycobacterium spp., Mycobacterium tuberculosis Complex, and Mycobacterium avium Complex Directly from Clinical Samples by Use of the BD Max Open System
Sonochemical synthesis and characterization of Pt/CNT, Pt/TiO2, and Pt/CNT/TiO2 electrocatalysts for methanol electro-oxidation
SSCI-VIDE+ECI2D+GBEInternational audienc
Schedule-Dependent Combined Sensitivity Testing of Anti-Cancer Agents in Human Gastric Carcinoma Cell Lines
Characteristics of De Novo Donor-specific Anti-HLA Antibodies (DSAs) in Living-donor Lobar and Cadaveric Lung Transplantation
Taq DNA Polymerase Mutants and 2′-Modified Sugar Recognition
Chemical modifications to DNA, such
as 2′ modifications,
are expected to increase the biotechnological utility of DNA; however,
these modified forms of DNA are limited by their inability to be effectively
synthesized by DNA polymerase enzymes. Previous efforts have identified
mutant <i>Thermus aquaticus</i> DNA polymerase I (Taq) enzymes
capable of recognizing 2′-modified DNA nucleotides. While these
mutant enzymes recognize these modified nucleotides, they are not
capable of synthesizing full length modified DNA; thus, further engineering
is required for these enzymes. Here, we describe comparative biochemical
studies that identify useful, but previously uncharacterized, properties
of these enzymes; one enzyme, SFM19, is able to recognize a range
of 2′-modified nucleotides much wider than that previously
examined, including fluoro, azido, and amino modifications. To understand
the molecular origins of these differences, we also identify specific
amino acids and combinations of amino acids that contribute most to
the previously evolved unnatural activity. Our data suggest that a
negatively charged amino acid at 614 and mutation of the steric gate
residue, E615, to glycine make up the optimal combination for modified
oligonucleotide synthesis. These studies yield an improved understanding
of the mutational origins of 2′-modified substrate recognition
as well as identify SFM19 as the best candidate for further engineering,
whether via rational design or directed evolution