Two-site ionic labeling with pyranine: implications for structural dynamics studies of polymers and polypeptides by time-resolved fluorescence anisotropy

Time-resolved fluorescence anisotropy (TRFA) is widely used to study dynamic motions of biomolecules in a variety of environments. However, depolarization due to rapid side chain motions often complicates the interpretation of anisotropy decay data and interferes with the accurate observation of segmental motions. Here, we demonstrate a new method for two-point ionic labeling of polymers and biomolecules that have appropriately spaced amino groups using the fluorescent probe 8-hydroxyl-1,3,6-trisulfonated pyrene (pyranine). TRFA analysis shows that such labeling provides a more rigid attachment of the fluorophore to the macromolecule than the covalent or single-point ionic labeling of amino groups, leading to time-resolved anisotropy decays that better reflect the backbone motion of the labeled polymer segment. Optimal coupling of pyranine to biomolecule dynamics is shown to be obtained for appropriately spaced Arg groups, and in such cases the ionic binding is stable up to 150 mM ionic strength. TRFA was used to monitor the behavior of pyranine-labeled poly(allylamine) (PAM) and poly-d-lysine (PL) in sodium silicate derived sol-gel materials and revealed significant restriction of backbone motion upon entrapment for both polymers, an observation that was not readily apparent in a previous study with entrapped fluorescein-labeled PAM and PL. The implications of these findings for fluorescence studies of polymer and biomolecule dynamics are discussed.     

An experimental switchable stiffness device for shock isolation

The development of an experimental switching stiffness device for shock isolation is presented. The system uses magnetic forces to exert a restoring force, which results in an effective stiffness that is used to isolate a payload. When the magnetic force is turned on and off, a switchable stiffness is obtained. Characterization of the physical properties of the device is presented. They are estimated in terms of the percentage stiffness change and effective damping ratio when switched between two constant stiffness states. Additionally, the setup is used to implement a control strategy to reduce the shock response and minimize residual vibration. The system was found to be very effective for shock isolation. The response is reduced by around 50 percent compared with passive isolation showing good correlation with theoretical predictions, and the effective damping ratio in the system following the shock was increased from about 4.5 percent to 13 percent.     

A Rapid Sputum-based Lateral Flow Assay for Airway Eosinophilia using an RNA-cleaving DNAzyme Selected for Eosinophil Peroxidase

The first protein-binding allosteric RNA-cleaving DNAzyme (RCD) obtained by direct in vitro selection against eosinophil peroxidase (EPX), a validated marker for airway eosinophilia, is described. The RCD has nanomolar affinity for EPX, shows high selectivity against related peroxidases and other eosinophil proteins, and is resistant to degradation by mammalian nucleases. An optimized RCD was used to develop both fluorescence and lateral flow assays, which were evaluated using 38 minimally processed patient sputum samples (23 non-eosinophilic, 15 eosinophilic), producing a clinical sensitivity of 100 % and specificity of 96 %. This RCD-based lateral flow assay should allow for rapid evaluation of airway eosinophilia as an aid for guiding asthma therapy.