Studies on the structure-function relationships and regulation mechanisms of Escherichia coli adenylosuccinate synthetase

Molecular biological, biochemical, and biophysical techniques were utilized to explore structure-function relationship of Escherichia coli adenylosuccinate synthetase. For the first time, residues Arg303, Arg304 and Arg305 were implicated as the determinants in the recognition and binding of substrate L-aspartate. Replacing Arg305 with leucine resulted in over a 100-fold decrease in kcat, suggesting a possible role of Arg305 in catalysis. pH dependent kinetic studies indicated that the side chain of Gln224 is not only involved in recognition of IMP, but also in stabiling the intermediate 6-phosphoryl-IMP. Substitution of the side chain amide with a carboxylate group resulted in loss of catalytic activity. The enzymes with mutations on the interface region, R143K, R1431, and D231A, along with wild type AMPSase, were analyzed using MALDI mass spectroscopy, sedimentation equilibrium, and initial rate kinetics. Ligand-induced dimerization is suggested as the regulatory mechanism for AMPSase, with the monomer as the inactive or lower active species, with the dimer as the fully active form;The purA gene was subcloned into the prokaryotic expression vector pTrc99A for better expression. The new construct pTrpA produced a 10- to 20-fold increase in enzyme yield compared to the older expression vector PMS204

Facile and Label-Free Electrochemical Biosensors for MicroRNA Detection based on DNA Origami Nanostructures

MicroRNAs (miRNAs) have emerged as the promising molecular biomarkers for early diagnosis and enhanced understanding of the molecular pathogenesis of cancers as well as certain diseases. Here, a facile, label-free, and amplification-free electrochemical biosensor was developed to detect miRNA by using DNA origami nanostructure-supported DNA probes, with methylene blue (MB) serving as the hybridization redox indicator, for the first time. Specifically, the use of cross-shaped DNA origami nanostructures containing multiple single-stranded DNA probes at preselected locations on each DNA nanostructure could increase the accessibility and the recognition efficiency of the probes (due to the rational controlled density of DNA probes). The successful immobilization of DNA origami probes and their hybridization with targeted miRNA-21 molecules was confirmed by electrochemical impedance spectroscopy and cyclic voltammetry methods. A differential pulse voltammetry technique was employed to record the oxidation peak current of MB before and after target hybridization. The linear detection range of this biosensor was from 0.1 pM to 10.0 nM, with a lower detection limit of 79.8 fM. The selectivity of the miRNA biosensor was also studied by observing the discrimination ability of single-base mismatched sequences. Because of the larger surface area and unprecedented customizability of DNA origami nanostructures, this strategy demonstrated great potential for sensitive, selective, and label-free determination of miRNA for translational biomedical research and clinical applications