The role of DNA nanostructures in the catalytic properties of an allosterically regulated protease.

DNA-scaffolded enzymes typically show altered kinetic properties; however, the mechanism behind this phenomenon is still poorly understood. We address this question using thrombin, a model of allosterically regulated serine proteases, encaged into DNA origami cavities with distinct structural and electrostatic features. We compare the hydrolysis of substrates that differ only in their net charge due to a terminal residue far from the cleavage site and presumably involved in the allosteric activation of thrombin.

Disulfide-Linked Allosteric Modulators for Multi-cycle Kinetic Control of DNA-Based Nanodevices.

Nature employs sulfur switches, that is, redox-active disulfides, to kinetically control biological pathways in a highly efficient and reversible way. Inspired by this mechanism, we describe herein a DNA-based synthetic nanodevice that acts as a sulfur switch and can be temporally controlled though redox regulation. To do this, we rationally designed disulfide DNA strands (modulators) that hybridize to a ligand-binding DNA nanodevice and act as redox-active allosteric regulators inducing the nanodevice to release or load its ligand.

A DNA-Based Biosensor Assay for the Kinetic Characterization of Ion-Dependent Aptamer Folding and Protein Binding.

Therapeutic and diagnostic nucleic acid aptamers are designed to bind tightly and specifically to their target. The combination of structural and kinetic analyses of aptamer interactions has gained increasing importance. Here, we present a fluorescence-based switchSENSE aptasensor for the detailed kinetic characterization of aptamer-analyte interaction and aptamer folding, employing the thrombin-binding aptamer (TBA) as a model system.