4 research outputs found
Validation of the Slow OffâKinetics of SirtuinâRearranging Ligands (SirReals) by Means of LabelâFree Electrically Switchable Nanolever Technologyâ
No full textWe have discovered the sirtuinârearranging ligands (SirReals) to be highly potent and selective inhibitors of the NAD+âdependent lysine deacetylase Sirt2. Using a biotinylated SirReal in combination with biolayer interferometry, we previously observed a slow dissociation rate of the inhibitorâenzyme complex; this had been postulated to be the key to the high affinity and selectivity of SirReals. However, to attach biotin to the SirReal core, we introduced a triazole as a linking moiety; this was shown by Xâray coâcrystallography to interact with Arg97 of the cofactor binding loop. Herein, we aim to elucidate whether the observed long residence time of the SirReals is induced mainly by triazole incorporation or is an inherent characteristic of the SirReal inhibitor core. We used the novel labelâfree switchSENSEÂź technology, which is based on electrically switchable DNA nanolevers, to prove that the long residence time of the SirReals is indeed caused by the core scaffold
Validation of Slow Off-Kinetics of Sirtuin Rearranging Ligands (SirReals) by Means of the Label-Free Electrically Switchable Nanolever Technology
No full textRecently, we have discovered the sirtuin rearranging ligands (SirReals) as a novel class of highly potent and selective inhibitors of the NAD+-dependent lysine deacetylase Sirt2. In previous studies, using a biotinylated SirReal analogue in combination with biolayer interferometry, we observed a slow dissociation rate of the inhibitor-enzyme complex, which had been postulated to be the key to the high affinity and selectivity of SirReals. However, for the attachment of biotin to the SirReal core, we introduced a triazole as a linking moiety, which was shown by X-ray co-crystallography to interact with Arg97 of the cofactor binding loop. This study now is directed to answer the question, whether the observed long residence time of the SirReals is induced mainly by triazole incorporation or is an inherent characteristic of the SirReal inhibitor core. Therefore, we used the novel label-free switchSENSEÂź technology, based on electrically switchable DNA nanolevers, to validate that the long residence time of the SirReals is caused by the core scaffold.<br /
A DNA-Based Biosensor Assay for the Kinetic Characterization of Ion-Dependent Aptamer Folding and Protein Binding
No full textTherapeutic 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
