RESUMO
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. Upon reduction, the allosteric modulator spontaneously de-hybridizes from the nanodevice and, as a result, its effect is transient. The system is reversible and has an unprecedented high tolerance to waste products and displays transient behavior for over 40â cycles without significant loss of efficiency. Kinetic control of DNA-based ligand-binding nanodevices through purely chemical reactions paves the way for temporal regulation of more complex chemical pathways.
Assuntos
DNA/metabolismo , Dissulfetos/metabolismo , Nanoestruturas/química , Nanotecnologia , Regulação Alostérica , DNA/química , Dissulfetos/química , CinéticaRESUMO
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. Thrombin-binding aptamer folding into a G-quadruplex and its binding to thrombin strongly depend on the type and concentration of ions present in solution. We observed conformational changes induced by cations in real-time and determined the folding and unfolding kinetics of the aptamer. The aptamer's affinity for K+ was found to be more than one order of magnitude higher than for other cations (K+ > NH4+ >> Na+ > Li+). The aptamer's affinity to its protein target thrombin in the presence of different cations followed the same trend but differed by more than three orders of magnitude (KD = 0.15 nM to 250 nM). While the stability (kOFF) of the thrombin-TBA complex was similar in all conditions, the cation type strongly influenced the association rate (kON). These results demonstrated that protein-aptamer binding is intrinsically related to the correct aptamer fold and, hence, to the presence of stabilizing ions. Because fast binding kinetics with on-rates exceeding 108 M-1s-1 can be quantified, and folding-related phenomena can be directly resolved, switchSENSE is a useful analytical tool for in-depth characterization of aptamer-ion and aptamer-protein interactions.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , DNA/química , Íons/química , Conformação de Ácido Nucleico , Proteínas/química , Quadruplex G , Cinética , Ligação Proteica , Proteínas/metabolismo , Trombina/químicaRESUMO
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. Our data show that the reaction rate is affected by DNA/substrate electrostatic interactions, proportionally to the degree of DNA/enzyme tethering. For substrates of opposite net charge, this leads to an inversion of the catalytic response of the DNA-scaffolded thrombin when compared to its freely diffusing counterpart. Hence, by altering the electrostatic environment nearby the encaged enzyme, DNA nanostructures interfere with charge-dependent mechanisms of enzyme-substrate recognition and may offer an alternative tool to regulate allosteric processes through spatial confinement.