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

The present work investigates the electrochemical determination of cocaine in the presence of levamisole, one of the most common adulterants found in cocaine street samples. Levamisole misleads cocaine color tests, giving a blue color (positive test) even in the absence of cocaine. Moreover, the electrochemical detection of cocaine is also affected by the presence of levamisole, with a suppression of the oxidation signal of cocaine. When levamisole is present in the sample in ratios higher than 1:1, the cocaine signal is no longer detected, thus leading to false negative results. Mass spectrometry and nuclear magnetic resonance were used to investigate if the signal suppression is due to the formation of a complex between cocaine and levamisole in bulk solution. Strategies to eliminate this suppressing effect are further suggested in this manuscript. In a first approach, the increase of the pH of the sample solution from pH 7 to pH 12 allowed the voltammetric determination of cocaine in the presence of levamisole in a concentration range from 10 to 5000 μM at nonmodified graphite disposable electrodes with a detection limit of 5 μM. In a second approach, the graphite electrode was cathodically pretreated, resulting in the presence of oxidation peaks of both cocaine and levamisole, with a detection limit for cocaine of 3 μM over the linear range of concentrations from 10 to 2500 μM. Both these strategies have been successfully applied for the simultaneous detection of cocaine and levamisole in three street samples on unmodified graphite disposable electrodes.

Rotihibins A and B are plant growth inhibitors acting on the TORK pathway. We report the isolation and characterization of new sequence analogues of rotihibin from Streptomyces scabies , a major cause of common scab in potato and other tuber and root vegetables.

Previous investigations of the impact of an imidazole-tethered thymidine in synthetic DNA duplexes, monitored using UV and NMR spectroscopy, revealed a base context dependent increase in thermal stability of these duplexes and a striking correlation with the imidazolium pKa. Unrestrained molecular dynamics (MD) simulations demonstrated the existence of a hydrogen bond between the imidazolium and the Hoogsteen side of a nearby guanosine which, together with electrostatic interactions, form the basis of the so-called pKa-motif responsible for these duplex-stabilizing and pKa-modulating properties. Here, the robustness and utility of this pKa-motif was explored by introducing multiple imidazole-tethered thymidines at different positions on the same dsDNA duplex. For all constructs, sequence based expectations as to pKa-motif formation were supported by MD simulations and experimentally validated using NOESY. Based on the analysis of the pKa values and melting temperatures, guidelines are formulated to assist in the rational design of oligonucleotides modified with imidazolium-tethered thymidines for increased thermal stability that should be generally applicable, as demonstrated through a triply modified construct. In addition, a proof-of-principle study demonstrating enhanced stability of the l-argininamide binding aptamer modified with an imidazole-tethered thymidine in the presence and absence of ligand, demonstrates its potential for the design of more stable aptamers.