No abstract

The efficiency of current microcystin detection methods has been hampered by the low detection limits required in drinking water and that routine detection is restricted to a few of the congeners with high degree of undesired cross-reactivity. Here, we report the development of novel microcystin-targeting molecules and their application in microcystin detection. We have selected DNA aptamers from a diverse random library that exhibit high affinity and specificity to microcystin-LR, -YR, and -LA. We obtained aptamers that bind to all chosen congeners with high affinity with K(D) ranging from 28 to 60 nM. More importantly, we also obtained aptamers that are selective among the different congeners, with selectivity from 3-folds difference in binding affinity to total discrimination (K(D) of 50 nM versus nonspecific binding). Electrochemical aptasensors constructed with the selected aptamers were able to achieve sensitive and congener-specific microcystin detection with detection limit as low as 10 pM.

No abstract

The Bacillus subtilis lysC lysine riboswitch modulates its own gene expression upon lysine binding through a transcription attenuation mechanism. The riboswitch aptamer is organized around a single five-way junction that provides the scaffold for two long-range tertiary interactions (loop L2–loop L3 and helix P2–loop L4)—all of this for the creation of a specific lysine binding site. We have determined that the interaction P2–L4 is particularly important for the organization of the ligand-binding site and for the riboswitch transcription attenuation control. Moreover, we have observed that a folding synergy between L2–L3 and P2–L4 allows both interactions to fold at lower magnesium ion concentrations. The P2–L4 interaction is also critical for the close juxtaposition involving stems P1 and P5. This is facilitated by the presence of lysine, suggesting an active role of the ligand in the folding transition. We also show that a previously uncharacterized stem–loop located in the expression platform is highly important for the riboswitch activity. Thus, folding elements located in the aptamer and the expression platform both influence the lysine riboswitch gene regulation.

No abstract

The paper presents for the first time a study of long-period gratings (LPGs) applied for label-free detection of specific bacteria using physically adsorbed bacteriophages. For the purposes of the experiment a number of highly sensitive LPGs working at the turning point of phase matching curve was fabricated in SMF28 fiber using UV exposure. We show that the device allows for real-time monitoring of phenomena taking place on the sensor's surface, including phage-bacteria interactions. For the applied conditions a resonance wavelength shift of ~1.3 nm induced by bacteria binding was observed.

Ligand binding to G protein-coupled receptors is a complex process that involves sequential receptor conformational changes, ligand translocation, and possibly ligand-induced receptor oligomerization. Binding events at muscarinic acetylcholine receptors are usually interpreted from radioligand binding studies in terms of two-step ligand-induced receptor isomerization. We report here, using a combination of fluorescence approaches, on the molecular mechanisms for Bodipypirenzepine binding to enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors in living cells. Real time monitoring, under steady-state conditions, of the strong fluorescence energy transfer signal elicited by this interaction permitted a fine kinetic description of the binding process. Timeresolved fluorescence measurements allowed us to identify discrete EGFP lifetime species and to follow their redistribution upon ligand binding. Fluorescence correlation spectroscopy, with EGFP brightness analysis, showed that EGFP-fused muscarinic M1 receptors predominate as monomers in the absence of ligand and dimerize upon pirenzepine binding. Finally, all these experimental data could be quantitatively reconciled into a three-step mechanism, with four identified receptor conformational states. Fast ligand binding to a peripheral receptor site initiates a sequence of conformational changes that allows the ligand to access to inner regions of the protein and drives ligandreceptor complexes toward a high affinity dimeric state. G protein-coupled receptors (GPCRs)3 trigger a wide palette of signaling pathways (1, 2), including G protein-independent responses (3). These receptors display multiple conformational and functional states, dependent on the cellular context, differentially selected and stabilized by ligands, and discriminated by downstream protein partners (4 -9). The occurrence of distinct receptor conformational species is supported by structural arguments provided by metal ion site engineering (10) or in situ disulfide cross-linking (11) and by direct monitoring of receptor intramolecular rearrangements through fluorescencebased methods (for reviews see Refs. 8,9,12).Few studies focused on the initial ligand binding step, its kinetic description, and its relationship with functionally relevant receptor conformational states. These aspects were addressed by monitoring intermolecular fluorescence resonance energy transfer (FRET) between a GFP-tagged receptor (donor) and a fluorescent ligand (acceptor). Both neurokinin A binding to class A tachykinin NK2 receptors (4, 13) and parathyroid hormone binding to class B parathyroid hormone receptors (14) proceeded in two steps, featuring two kinetically distinguishable conformational states. Whether such biphasic binding reactions are a general feature of GPCRs, independent on the pharmacological nature of the ligand, and whether they reflect different receptor functional states or sequential binding steps remain important questions to be elucidated.Muscarinic cholinergic receptors (15) disp...

No abstract