Since their inception, DNA aptamers were regarded as the turning point for biochemical sensing in real samples; however up to now their promises are far from being fulfilled. Especially aptamers for small molecules pose a challenge for both selection and characterization. The lack of a universally accepted and robust quality control protocol for the characterization of aptamer performances coupled with the observation of inconsistent data sets in literature, prompted us to address the issue comparing different analytical methodologies to validate (or disprove) the binding capabilities of aptamer sequences. We chose three aptamers for ampicillin, a β-Lactam antibiotic; used several detection strategies described in literature. The colorimetric gold nanoparticles (AuNPs) assay used in the original paper describing the aptamer sequences was repeated with conflicting results. The three sequences were then tested with three different instrumental techniques to assess their Kd and binding mechanism in homogeneous solutions. Coupling the thermodynamic data obtained with Isothermal Titration Calorimetry (ITC) with the structural information on the binding event given by Native Electro Spray Ionization Mass spectrometry (Native ESI-MS) and 1H-NMR it was possible to verify that the three sequences do not show any specific binding with the target ampicillin. To verify the influence of the AuNPs on the binding event, the experiments were repeated in presence of AuNPs both with ITC and 1H-NMR, again without any results. By offering a cross-referenced and robust analitycal approach to aptamer characterization we aim at elucidating the potentialities of aptamer for small organic molecules, especially when ultrasensitive analytical application are involved

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Perfluorooctanoic acid (PFOA) is a toxic compound that is absorbed and distributed throughout the body by noncovalent binding to serum proteins such as human serum albumin (hSA). Though the interaction between PFOA and hSA has been already assessed using various analytical techniques, a high resolution and detailed analysis of the binding mode is still lacking. We report here the crystal structure of hSA in complex with PFOA and a medium-chain saturated fatty acid (FA). A total of eight distinct binding sites, four occupied by PFOAs and four by FAs, have been identified. In solution binding studies confirmed the 4:1 PFOA-hSA stoichiometry and revealed the presence of one high and three low affinity binding sites. Competition experiments with known hSA-binding drugs allowed locating the high affinity binding site in subdomain IIIA. The elucidation of the molecular basis of the interaction between PFOA and hSA might provide not only a better assessment of the absorption and elimination mechanisms of these compounds in vivo but also have implications for the development of novel molecular receptors for diagnostic and biotechnological applications. K E Y W O R D S binding mode, crystal structure, fluoroalkyl substances, human serum albumin, molecular interaction, perfluorooctanoic acid 1 | INTRODUCTION Perfluorooctanoic acid (PFOA) is a perfluoroalkyl substance (PFAS) with a carboxyl functional group and seven fluorinated carbon atoms. 1 PFOA is a man-made

Mutations in the Parkinson's disease (PD)-associated protein leucine-rich repeat kinase 2 (LRRK2) commonly lead to a reduction of GTPase activity and increase in kinase activity. Therefore, strategies for drug development have mainly been focusing on the design of LRRK2 kinase inhibitors. We recently showed that the central RocCOR domains (Roc: Ras of complex proteins; COR: C-terminal of Roc) of a bacterial LRRK2 homolog cycle between a dimeric and monomeric form concomitant with GTP binding and hydrolysis. PD-associated mutations can slow down GTP hydrolysis by stabilizing the protein in its dimeric form. Here, we report the identification of two Nanobodies (NbRoco1 and NbRoco2) that bind the bacterial Roco protein (CtRoco) in a conformation-specific way, with a preference for the GTP-bound state. NbRoco1 considerably increases the GTP turnover rate of CtRoco and reverts the decrease in GTPase activity caused by a PD-analogous mutation. We show that NbRoco1 exerts its effect by allosterically interfering with the CtRoco dimer–monomer cycle through the destabilization of the dimeric form. Hence, we provide the first proof of principle that allosteric modulation of the RocCOR dimer–monomer cycle can alter its GTPase activity, which might present a potential novel strategy to overcome the effect of LRRK2 PD mutations.

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