ABSTRACT
The MutS2 homologues have received attention because of their unusual activities that differ from those of MutS. In this work, we report on the functional characteristics and conformational diversities of Thermotoga maritima MutS2 (TmMutS2). Various biochemical features of the protein were demonstrated via diverse techniques such as scanning probe microscopy (SPM), ATPase assays, analytical ultracentrifugation, DNA binding assays, size chromatography, and limited proteolytic analysis. Dimeric TmMutS2 showed the temperature-dependent ATPase activity. The non-specific nicking endonuclease activities of TmMutS2 were inactivated in the presence of nonhydrolytic ATP (ADPnP) and enhanced by the addition of TmMutL. In addition, TmMutS2 suppressed the TmRecA-mediated DNA strand exchange reaction in a TmMutL-dependent manner. We also demonstrated that small-angle X-ray scattering (SAXS) analysis of dimeric TmMutS2 exhibited nucleotide- and DNA-dependent conformational transitions. Particularly, TmMutS2-ADPnP showed the most compressed form rather than apo-TmMutS2 and the TmMutS2-ADP complex, in accordance with the results of biochemical assays. In the case of the DNA-binding complexes, the stretched conformation appeared in the TmMutS2-four-way junction (FWJ)-DNA complex. Convergences of biochemical- and SAXS analysis provided abundant information for TmMutS2 and clarified ambiguous experimental results.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Thermotoga maritima/enzymology , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , DNA Repair , DNA, Bacterial/genetics , Homologous Recombination , Hydrolases/chemistry , Hydrolases/metabolism , Models, Molecular , Protein Conformation , Scattering, Small Angle , Thermotoga maritima/chemistry , Thermotoga maritima/genetics , Thermotoga maritima/metabolism , X-Ray DiffractionABSTRACT
A polymer-based aptasensor, which consisted of fluorescein amidite (FAM)-modified aptamers and coordination polymer nanobelts (CPNBs), was developed utilizing the fluorescence quenching effect to detect sulfadimethoxine residue in food products. A single-stranded DNA (ssDNA) aptamer, which was a specific bio-probe for sulfadimethoxine (Su13; 5'-GAGGGCAACGAGTGTTTATAGA-3'), was discovered by a magnetic bead-based systematic evolution of ligands by exponential enrichment (SELEX) technique, and the fluorescent quenchers CPNBs were produced by mixing AgNO(3) and 4,4'-bipyridine. This aptasensor easily and sensitively detected sulfadimethoxine in solution with a limit of detection (LOD) of 10ng/mL. Furthermore, the antibiotic dissolved in milk was also effectively detected with the same LOD value. In addition, this aptamer probe offered high specificity for sulfadimethoxine compared to other antibiotics. These valuable results provide ample evidence that the CPNB-based aptasensor can be used to quantify sulfadimethoxine residue in food products.
Subject(s)
Anti-Infective Agents/analysis , Biosensing Techniques/methods , SELEX Aptamer Technique , Sulfadimethoxine/analysis , Animals , Base Sequence , Fluorescence , Limit of Detection , Milk/chemistry , Molecular Sequence DataABSTRACT
A gold nanoparticle based dual fluorescence-colorimetric method was developed as an aptasensor to detect ampicillin using its single-stranded DNA (ssDNA) aptamer, which was discovered by a magnetic bead-based SELEX technique. The selected aptamers, AMP4 (5'-CACGGCATGGTGGGCGTCGTG-3'), AMP17 (5'-GCGGGCGGTTGTATAGCGG-3'), and AMP18 (5'-TTAGTTGGGGTTCAGTTGG-3'), were confirmed to have high sensitivity and specificity to ampicillin (K(d), AMP7 = 9.4 nM, AMP17 = 13.4 nM, and AMP18 = 9.8 nM, respectively). The 5'-fluorescein amidite (FAM)-modified aptamer was used as a dual probe for observing fluorescence differences and color changes simultaneously. The lower limits of detection for this dual method were a 2 ng/mL by fluorescence and a 10 ng/mL by colorimetry for ampicillin in the milk as well as in distilled water. Because these detection limits were below the maximum residue limit of ampicillin, this aptasensor was sensitive enough to detect antibiotics in food products, such as milk and animal tissues. In addition, this dual aptasensor will be a more accurate method for antibiotics in food products as it concurrently uses two detection methods: fluorescence and colorimetry.
