Comparison of Solution and Crystal Structures of PreQ₁ Riboswitch Reveals Calcium-Induced Changes in Conformation and Dynamics

Riboswitches regulate gene expression via specific recognition of cognate metabolites by their aptamer domains, which fold into stable conformations upon ligand binding. However, the recently reported solution and crystal structures of the Bacillus subtilis preQ₁ riboswitch aptamer show small but significant differences, suggesting that there may be conformational heterogeneity in the ligand-bound state. We present a structural and dynamic characterization of this aptamer by solution NMR spectroscopy. The aptamer−preQ₁ complex is intrinsically flexible in solution, with two regions that undergo motions on different time scales. Three residues move in concert on the micro-to-millisecond time scale and may serve as the lid of the preQ₁-binding pocket. Several Ca²⁺ ions are present in the crystal structure, one of which binds with an affinity of 47 ± 2 μM in solution to a site that is formed only upon ligand binding. Addition of Ca²⁺ to the aptamer−preQ₁ complex in solution results in conformational changes that account for the differences between the solution and crystal structures. Remarkably, the Ca²⁺ ions present in the crystal structure, which were proposed to be important for folding and ligand recognition, are not required for either in solution

Electrotriggered, Spatioselective, Quantitative Gene Delivery into a Single Cell Nucleus by Au Nanowire Nanoinjector

Delivery of bioactive materials into a cell is highly important in the study of cell biology and medical treatments. Ideal nanoinjectors should be able to deliver biomaterials with high spatial resolution while causing minimum cell damage. We developed a Au nanowire (NW) nanoinjector that has the thinnest diameter (100–150 nm) among the DNA delivering devices as well as optimum mechanical properties, minimizing cell damage. Well-defined (111) single-crystalline Au surface and high electric conductivity of a Au NW nanoinjector allow precisely timed and efficient electrochemical release of DNA molecules attached on a Au NW surface. Both linear DNA and plasmid DNA were delivered separately and showed successful expression. The Au NW nanoinjector would find important biomedical applications in the fields such as gene therapy, DNA vaccination, targeted drug delivery, and probe/control of cell signaling events

Regenerative Astaxanthin Extraction from a Single Microalgal (<i>Haematococcus pluvialis</i>) Cell Using a Gold Nano-Scalpel

Milking of microalgae, the process of reusing the biomass for continuous production of target compounds, can strikingly overcome the time and cost constraints associated with biorefinery. This process can significantly improve production efficiency of highly valuable chemicals, for example, astaxanthin (AXT) from Haematococcus pluvialis. Detailed understanding of the biological process of cell survival and AXT reaccumulation after extraction would be of great help for successful milking. Here we report extraction of AXT from a single cell of H. pluvialis through incision of the cell wall by a gold nanoscalpel (Au-NS), which allows single-cell analysis of wound healing and reaccumulation of AXT. Interestingly, upon the Au-NS incision, the cell could reaccumulate AXT at a rate two times faster than the control cells. Efficient extraction as well as minimal cellular damage, keeping cells alive, could be achieved with the optimized shape and dimensions of Au-NS: a well-defined sharp tip, thickness under 300 nm, and 1–3 μm of width. The demonstration of regenerative extraction of AXT at a single cell level hints toward the potential of a milking process for continuous recovery of target compounds from microalgae while keeping the cells alive

Subcellular Neural Probes from Single-Crystal Gold Nanowires

Size reduction of neural electrodes is essential for improving the functionality of neuroprosthetic devices, developing potent therapies for neurological and neurodegenerative diseases, and long-term brain–computer interfaces. Typical neural electrodes are micromanufactured devices with dimensions ranging from tens to hundreds of micrometers. Their further miniaturization is necessary to reduce local tissue damage and chronic immunological reactions of the brain. Here we report the neural electrode with subcellular dimensions based on single-crystalline gold nanowires (NWs) with a diameter of ∼100 nm. Unique mechanical and electrical properties of defect-free gold NWs enabled their implantation and recording of single neuron-activities in a live mouse brain despite a ∼50× reduction of the size compared to the closest analogues. Reduction of electrode dimensions enabled recording of neural activity with improved spatial resolution and differentiation of brain activity in response to different social situations for mice. The successful localization of the epileptic seizure center was also achieved using a multielectrode probe as a demonstration of the diagnostics potential of NW electrodes. This study demonstrated the realism of single-neuron recording using subcellular-sized electrodes that may be considered a pivotal point for use in diverse studies of chronic brain diseases

Redox Probing for Chemical Information of Oxidative Stress

Oxidative stress is implicated in many diseases yet no simple, rapid, and robust measurement is available at the point-of-care to assist clinicians in detecting oxidative stress. Here, we report results from a discovery-based research approach in which a redox mediator is used to probe serum samples for chemical information relevant to oxidative stress. Specifically, we use an iridium salt (K₂IrCl₆) to probe serum for reducing activities that can transfer electrons to iridium and thus generate detectable optical and electrochemical signals. We show that this Ir-reducing assay can detect various biological reductants and is especially sensitive to glutathione (GSH) compared to alternative assays. We performed an initial clinical evaluation using serum from 10 people diagnosed with schizophrenia, a mental health disorder that is increasingly linked to oxidative stress. The measured Ir-reducing capacity was able to discriminate people with schizophrenia from healthy controls (<i>p</i> < 0.005), and correlations were observed between Ir-reducing capacity and independent measures of symptom severity

Radical Scavenging Activities of Biomimetic Catechol-Chitosan Films

Recent studies showed that melanin-mimetic catechol-chitosan films are redox-active and their ability to exchange electrons confers pro-oxidant activities for the sustained, <i>in situ</i> generation of reactive oxygen species for antimicrobial bandages. Here we electrofabricated catechol-chitosan films, demonstrate these films are redox-active, and show their ability to exchange electrons confers sustained radical scavenging activities that could be useful for protective coatings. Electrofabrication was performed in two steps: cathodic electrodeposition of a chitosan film followed by anodic grafting of catechol to chitosan. Spectroelectrochemical reverse engineering methods were used to characterize the catechol-chitosan films and demonstrate the films are redox-active and can donate electrons to quench oxidative free radicals and can accept electrons to quench reductive free radicals. Electrofabricated catechol-chitosan films that were peeled from the electrode were also shown to be capable of donating electrons to quench an oxidative free radical, but this radical scavenging activity decayed upon depletion of electrons from the film (i.e., as the film became oxidized). However, the radical scavenging activity could be recovered by a regeneration step in which the films were contacted with the biological reducing agent ascorbic acid. These results demonstrate that catecholic materials offer important redox-based and context-dependent properties for possible applications as protective coatings