Measurement of (Aptamer–Small Target) <i>K</i>_D Using the Competition between Fluorescently Labeled and Unlabeled Targets and the Detection of Fluorescence Anisotropy

Registration of fluorescence anisotropy (FA) allows for characterizing the interactions of ligands with aptamers and other receptors under homogeneous conditions without reagent immobilization, prolonged incubations, and product separation. We proposed an approach for aptamer affinity determination by FA taking into account the difference in label fluorescence before and after complexation. The detailed step by step scheme using a native and fluorescently labeled ligand was described and justified in the paper. The scheme ensures the exclusion of data with low reliability and establishes valid criteria for selecting optimal concentrations of reagents (labeled ligand and aptamer) used in the experiments. The approach was experimentally tested using ochratoxin A (OTA), its fluorescein-labeled derivative (OTA-Flu), and the aptamer binding them. We demonstrated that it allows minimizing the influence of fluorescence change to accurately determine the dissociation constant. On the basis of FA registration, the binding constants of the aptamer–OTA-Flu and the aptamer–OTA complexes were found to be equal to 245 + 33 and 63 + 18 nM, respectively. The value for the aptamer–OTA complexes was confirmed by the equilibrium dialysis technique. The resulting constant was 80 ± 9 nM. The versatility and methodological simplicity of the proposed protocol, as well as the short implementation time, are why it can be recommended as an effective tool for characterizing aptamer–ligand complexes

Silver-enhanced lateral flow immunoassay for highly-sensitive detection of potato leafroll virus

<p>Rapid non-laboratory screening of plants for pathogenic viruses crucially influences crop yields in modern agricultural technologies. The aim of this study was to develop a highly-sensitive lateral flow immunoassay (LFIA) for rapid detection of potato leafroll virus (PLRV), an infectious agent of one of the most widespread potato diseases. The proposed LFIA combines the formation of sandwich immune complexes with gold nanoparticles (GNP) as labels and silver enhancement. The enhancement stage was realized using mixture of silver lactate and hydroquinone and subsequent addition of chloride-containing buffer to stop silver reduction. LFIA with silver enhancement was 15 times more sensitive (detection limit 0.2 ng/mL; 15 min) compared with conventional LFIA (detection limit 3 ng/mL; 10 min). The enhanced LFIA detected PLRV in leaves’ extracts of infected potato in dilutions higher than enzyme-linked immunosorbent assay.</p

Monomeric 14-3-3ζ Has a Chaperone-Like Activity and Is Stabilized by Phosphorylated HspB6

Members of the 14-3-3 eukaryotic protein family predominantly function as dimers. The dimeric form can be converted into monomers upon phosphorylation of Ser⁵⁸ located at the subunit interface. Monomers are less stable than dimers and have been considered to be either less active or even inactive during binding and regulation of phosphorylated client proteins. However, like dimers, monomers contain the phosphoserine-binding site and therefore can retain some functions of the dimeric 14-3-3. Furthermore, 14-3-3 monomers may possess additional functional roles owing to their exposed intersubunit surfaces. Previously we have found that the monomeric mutant of 14-3-3ζ (14-3-3ζ_m), like the wild type protein, is able to bind phosphorylated small heat shock protein HspB6 (pHspB6), which is involved in the regulation of smooth muscle contraction and cardioprotection. Here we report characterization of the 14-3-3ζ_m/pHspB6 complex by biophysical and biochemical techniques. We find that formation of the complex retards proteolytic degradation and increases thermal stability of the monomeric 14-3-3, indicating that interaction with phosphorylated targets could be a general mechanism of 14-3-3 monomers stabilization. Furthermore, by using myosin subfragment 1 (S1) as a model substrate we find that the monomer has significantly higher chaperone-like activity than either the dimeric 14-3-3ζ protein or even HspB6 itself. These observations indicate that 14-3-3ζ and possibly other 14-3-3 isoforms may have additional functional roles conducted by the monomeric state