Structure and thickness dependence of "molecular wiring" in nanostructured enzyme multilayers.

Supramolecular organized multilayers composed of glucose oxidase (GOx) and osmium-derivatized poly(allylamine) redox polymer have been self-assembled electrostatically from Os-polyelectrolyte solutions of variable pH (5.5-8.8) leading to a decrease of the linear charge density in the PAH-Os with increasing pH. The layer-by-layer enzyme multilayers were studied by ellipsometry, quartz crystal microbalance, AFM, cyclic voltammetry, and electrocatalytic oxidation of beta-D-glucose. At higher adsorption solution pH, an increase in the film thickness, enzyme loading, and redox charge was observed. While the electrocatalytic response increases with the increase of the adsorption solution pH (decrease of the polyelectrolyte linear charge), the FADH2 oxidation bimolecular rate constant has a maximum in the pH range 7.0-7.5 where a change in the film growth mechanism is observed.

Nanoscale memory provided by thermoreversible stochastically structured polymer aggregates on mica.

Stimuli-responsive polymers are used in a large variety of applications due to the controlled manner in which their physical properties can be reversibly altered. In this study, we demonstrate the thermoreversible structuring of poly(N-isopropylacrylamide)-based polymer. By temperature-controlled atomic force microscopy, we demonstrate that polymer aggregates form on mica above the polymer lower critical solution temperature and disperse below it, and in so doing, display positional "memory" in that the nanodomains are retained in the same positions and with the same shapes during repeated cooling/heating cycles. Such positional "memory" may be useful for multiple applications in nano-microscale devices.

Long-range RNA-RNA interactions circularize the dengue virus genome.

Secondary and tertiary RNA structures present in viral RNA genomes play essential regulatory roles during translation, RNA replication, and assembly of new viral particles. In the case of flaviviruses, RNA-RNA interactions between the 5' and 3' ends of the genome have been proposed to be required for RNA replication. We found that two RNA elements present at the ends of the dengue virus genome interact in vitro with high affinity. Visualization of individual molecules by atomic force microscopy revealed that physical interaction between these RNA elements results in cyclization of the viral RNA. Using RNA binding assays, we found that the putative cyclization sequences, known as 5' and 3' CS, present in all mosquito-borne flaviviruses, were necessary but not sufficient for RNA-RNA interaction. Additional sequences present at the 5' and 3' untranslated regions of the viral RNA were also required for RNA-RNA complex formation. We named these sequences 5' and 3' UAR (upstream AUG region). In order to investigate the functional role of 5'-3' UAR complementarity, these sequences were mutated either separately, to destroy base pairing, or simultaneously, to restore complementarity in the context of full-length dengue virus RNA. Nonviable viruses were recovered after transfection of dengue virus RNA carrying mutations either at the 5' or 3' UAR, while the RNA containing the compensatory mutations was able to replicate. Since sequence complementarity between the ends of the genome is required for dengue virus viability, we propose that cyclization of the RNA is a required conformation for viral replication.