Single molecule studies of rna-target interactions

We have used FRET, single molecule spectroscopy, and several other biophysical techniques to study how the folding of RNA allows it to perform its various functions by recognizing and binding a target ligand. We have shown that the c-di-GMP riboswitch undergoes a large, global conformational change upon binding of the ligand. The folding dynamics of the c-di-GMP riboswitch upon Mg2+ binding help to pre-organize the aptamer for efficient ligand binding and ultimately efficient gene expression. We have also investigated two instances, a fluorophore-binding aptamer and molecular beacon, where the folding of RNA can be used to detect an analyte. We have demonstrated that binding of the fluorophore, TAMRA, to an aptamer enhances the fluorescent properties of the fluorophore. Specifically, it increases both its quantum yield and fluorescent lifetime. This will provide an added advantage to the use of these aptamers in labeling an mRNA in vivo. We have shown that 2\u27 modifications do not affect the folding and binding of a molecular beacon to its target. The common use of these 2\u27 modifications does not hamper their use for in vivo studies. Whether natural or artificial, the folding of RNA to specifically recognize a target can be employed by both cells and scientists to perform a wide array of functions

Allosteric Tertiary Interactions Preorganize the c-di-GMP Riboswitch and Accelerate Ligand Binding

Cyclic diguanylate (c-di-GMP) is a bacterial second messenger important for physiologic adaptation and virulence. Class-I c-di-GMP riboswitches are phylogenetically widespread and thought to mediate pleiotropic genetic responses to the second messenger. Previous studies suggest that the RNA aptamer domain switches from an extended free state to a compact, c-di-GMP-bound conformation in which two helical stacks dock side-by-side. Single molecule fluorescence resonance energy transfer (smFRET) experiments now reveal that the free RNA exists in four distinct populations that differ in dynamics in the extended and docked conformations. In the presence of c-di-GMP and Mg²⁺, a stably docked population (>30 min) becomes predominant. smFRET mutant analysis demonstrates that tertiary interactions distal to the c-di-GMP binding site strongly modulate the RNA population structure, even in the absence of c-di-GMP. These allosteric interactions accelerate ligand recognition by preorganizing the RNA, favoring rapid c-di-GMP binding

Cooperative Prevention Systems to Protect Rangelands From the Spread of Invasive Plants

Eastern Montana is a vast region dominated by weed-free plains grassland. The protection of these prairies from the spread of invasive plants through weed prevention areas (WPAs) can preserve highquality rangelands for wildlife, livestock, and other ecosystem goods and services. Eastern Montana sustains a spacious region of native grasslands and shrublands (approximately 57,000 mi2). The conservation of these rangelands has farreaching societal implications for long-term sustainability of ecosystem services and rural livelihoods. Fortunately, most rangelands in eastern Montana still remain largely free of invasive weeds, or non-native plants that invade ecosystems and replace native plants. 1 Preventing their spread into rangelands is critical because invasive weeds can alter ecosystem function, reduce habitat and grazing land, and cause damaging economic effects for land managers. 2,3 Rangelands are susceptible to invasion due to frequent openings in plant cover, high light levels, and human activity that transports weeds and creates disturbance. Proactive strategies to counter invasion can keep rangelands weed-free, reducing the risk of environmental degradation, an outcome that could prove excessively costly to repair. Our objective is to illustrate the development and design of WPAs and the critical weed prevention actions that were developed in 12 WPAs in eastern Montana. The protection of weed-free prairie yields societal benefits that are a public good whereby everyone benefits from a healthy environment. A large part of the prevention task, however, is convincing the public that although weed spread displays slow moving trends, and thus no obvious threats, it is a long-term crisis worthy of our best efforts. Prevention is promoted through local campaigns including 1) weed-free ranch signage and roadside kiosks designating WPAs, 2) messaging and monthly prevention tips to ranchers via local newspapers, 3) field tours to sites dominated by weeds, 4) passive monitoring networks, and 5) prevention symposiums to share information and encourage collaboration between researchers and land managers

Global Analysis of Perovskite Photophysics Reveals Importance of Geminate Pathways

Hybrid organic–inorganic perovskites demonstrate desirable photophysical behaviors and promising applications from efficient photovoltaics to lasing, but the fundamental nature of excited state species is still under debate. We collected time-resolved photoluminescence of single-crystal nanoplates of methylammonium lead iodide perovskite (MAPbI₃) with excitation over a range of fluences and repetition rates to provide a more complete photophysical picture. A fundamentally different way of simulating the photophysics is developed that relies on unnormalized decays, global analysis over a large array of conditions, and inclusion of steady-state behavior; these details are critical to capturing observed behaviors. These additional constraints require inclusion of spatially correlated pairs along with free carriers and traps, demonstrating the importance of our comprehensive analysis. Modeling geminate and nongeminate pathways shows that geminate processes are dominant at high carrier densities and early times and that geminate recombination is catalyzed by free holes. Our combination of data and simulation provides a detailed picture of perovskite photophysics across multiple excitation regimes that was not previously available

Tracking Lithium Ions via Widefield Fluorescence Microscopy for Battery Diagnostics

Direct tracking of lithium ions with time and spatial resolution can provide an important diagnostic tool for understanding mechanisms in lithium ion batteries. A fluorescent indicator of lithium ions, 2-(2-hydroxyphenyl)­naphthoxazole, was synthesized and used for real-time tracking of lithium ions via widefield fluorescence microscopy. The fluorophore can be excited with visible light and was shown to enable quantitative determination of the lithium ion diffusion constant in a microfluidic model system for a plasticized polymer electrolyte lithium battery. The use of widefield fluorescence microscopy for in situ tracking of lithium ions in batteries is discussed

Vasopressin does not mediate hypersensitivity of the hypothalamic pituitary adrenal axis during chronic stress

The hypothesis that vasopressin (VP) becomes the main mediator of pituitary corticotroph responsiveness during chronic hypothalamic pituitary adrenal (HPA) activation, was tested by examining the effect of pharmacologic VP receptor blockade on the ACTH and corticosterone responses of 14-day repeatedly restrained rats. In spite of the increased vasopressinergic activity, repeatedly restrained rats showed lower ACTH and corticosterone responses to 10 min white noise compared with handled controls. These responses were unchanged by the non-peptide selective V1b receptor antagonist, SSR149415, i.v., 1h before noise application. In contrast to noise stress, plasma ACTH responses to i.p. hypertonic saline injection were enhanced in the repeatedly restrained rats compared with handled controls but responses were also unaffected by SSR149415 administered orally, daily 1 h before restraint. Since SSR149415 effectiveness was low, we used minipump infusion of the peptide V1 receptor antagonist, dGly[Phaa1,D-Tyr(et), Lys, Arg]VP (V1-Ant) for 14 days, which effectively blocked ACTH responses to exogenous VP. Chronic V1-Ant infusion reduced plasma ACTH responses to i.p. hypertonic saline in handled controls but not in repeatedly restrained rats. These data suggest that the increased vasopressinergic activity characteristic of chronic stress plays roles other than mediating the hypersensitivity of the HPA axis to a novel stress