Oxidation of guanines in the iron-responsive element RNA: similar structures from chemical modification and recent NMR studies

AbstractBackground: The translation or stability of the mRNAs from ferritin, m-aconitase, erythroid aminoevulinate synthase and the transferrin receptor is controlled by the binding of two iron regulatory proteins to a family of hairpin-forming RNA sequences called iron-responsive elements (IREs). The determination of higher-solution nuclear magnetic resonance (NMR) structures of IRE variants suggests an unusual hexaloop structure, leading to an intra-loop G-C base pair and a highly exposed loop guanine, and a special internal loop/bulge in the ferritin IRE involving a shift in base pairing not predicted with standard algorithms.Results: Cleavage of synthetic 55- and 30-mer RNA oligonucleotides corresponding to the ferritin IRE with complexes based on oxoruthenium(IV) shows enhanced reactivity at a hexaloop guanine and at a guanine adjacent to the internal loop/bulge with strong protection at a guanine in the internal loop/bulge. These results are consistent with the recent NMR structures. The synthetic 55-mer RNA binds the iron-regulatory protein from rabbit reticulocyte lysates. The DNA analogs of the 55- and 30-mers do not show the same reactivity pattern.Conclusions: The chemical reactivity of the guanines in the ferritin IRE towards oxoruthenium(IV) supports the published NMR structures and the known oxidation chemistry of the metal complexes, The results constitute progress towards developing stand-alone chemical nucleases that reveal significant structural properties and provide results that can ultimately be used to constrain molecular modeling

Nature of Guanine Oxidation in RNA via the Flash-Quench Technique versus Direct Oxidation by a Metal Oxo Complex

Oxidation of RNA can be effected by two different techniques: a photochemical, electron-transfer method termed “flash-quench” and direct oxidation by metal oxo complexes. The flash-quench method produces selective oxidation using a metal photosensitizer, tris(bipyridyl)ruthenium(III) trichloride (Ru(bpy)33+), and quencher, pentaamminechlorocobalt(III) chloride (Co(NH3)5Cl2+). We have optimized the flash-quench technique for the following RNAs: tRNAPhe, human ferritin iron-responsive element (IRE), and a mutated human ferritin IRE. We have also employed a chemical footprinting technique involving the oxoruthenium(IV) complex (Ru(tpy)(bpy)O2+ (tpy = 2,2′,2″-terpyridine; bpy = 2,2′-bipyridine)) to oxidize guanine. Comparison of the two methods shows that the flash-quench technique provides a visualization of nucleotide accessibility for a static conformation of RNA while the Ru(tpy)(bpy)O2+ complex selectively oxidizes labile guanines and gives a visualization of a composite of multiple conformations of the RNA structure

Using scenarios to explore UK upland futures

Uplands around the world are facing significant social, economic and environmental changes, and decision-makers need to better understand what the future may hold if they are to adapt and maintain upland goods and services. This paper draws together all major research comprising eight studies that have used scenarios to describe possible futures for UK uplands. The paper evaluates which scenarios are perceived by stakeholders to be most likely and desirable, and assesses the benefits and drawbacks of the scenario methods used in UK uplands to date. Stakeholders agreed that the most desirable and likely scenario would be a continuation of hill farming (albeit at reduced levels) based on cross-compliance with environmental measures. The least desirable scenario is a withdrawal of government financial support for hill farming. Although this was deemed by stakeholders to be the least likely scenario, the loss of government support warrants close attention due to its potential implications for the local economy. Stakeholders noted that the environmental implications of this scenario are much less clear-cut. As such, there is an urgent need to understand the full implications of this scenario, so that upland stakeholders can adequately prepare, and policy-makers can better evaluate the likely implications of different policy options. The paper concludes that in future, upland scenario research needs to: (1) better integrate in-depth and representative participation from stakeholders during both scenario development and evaluation; and (2) make more effective use of visualisation techniques and simulation models

The Photochemistry of Dioxorhenium(V)

The excited-state properties of trans-ReO2(py)4+ (ReO2+) in acetonitrile solution have been investigated. The excited-state absorption spectrum of ReO2+ is dominated by bleaching of the ground state MLCT and d-d systems. The reduction potential of ReO22+/+* is estimated from emission and electrochemical data to be -0.7 V (SSCE). The ReO2+ excited state efficiently reduces methylviologen and other pyridinium and olefin acceptors. The resulting Re(VI) species oxidizes secondary alcohols and silanes. Acetophenone is the product of sec-phenethyl alcohol oxidation. The emission properties of ReO2+ in aqueous solutions of anionic and nonionic surfactants have been investigated. The emission and absorption maxima of ReO2+ are dependent on the water content of its environment. Emission lifetimes vary over four orders of magnitude upon shifting from aqueous to nonaqueous environments. The emission lifetime has a large (8.6) isotope effect (k(H2O)/k(D2O)) that reflects its sensitivity towards the environment. These properties have been used to develop a model for the interactions of ReO2+ with sodium dodecyl sulfate (SDS). A hydrophobic ReO2+ derivative, ReO2(3-Ph-py)4+, has been used to probe micelles of nonionic surfactants, and these results are consistent with those obtained with SDS. The emission properties of ReO2+ in Nafion perfluorosulfonated membranes have been investigated. Absorption and emission spectroscopy indicate that the interior of the membrane is quite polar, similar to ethylene glycol. Two well-resolved emission components show different lifetimes and different isotope effects, indicative of varying degrees of solvent accessibility. These components are taken as evidence for chemically distinct regions in the polymer film, assigned as the interfacial region and the ion cluster region. The unsubstituted pyridine complex shows monophasic, τ = 1.7 µs, emission decay when bound to calf thymus DNA. Switching to the 3-Ph-py complex yields a biphasic emission decay (τ1 = 2.4 µs, τ2 = 10 µs) indicative of an additional, solvent-inaccessible binding mode. Photoinduced electron transfer to methylviologen leads to oxidative cleavage of the DNA as detected by gel electrophoresis. Electrochemical and spectrophotometric techniques used with organic substrates also can be used to monitor the oxidation of DNA. Abstraction of the ribose 4' hydrogen by ReO22+ is a possible mechanism.</p

Distribution of Metal Complexes Bound to DNA Determined by Normal Pulse Voltammetry

The effects of DNA binding on the normal pulse voltammetry of metal complexes have been investigated. Studies were performed both for oxidation of OsL_3^(2+/3+) and for reduction of CoL_3^(3+/2+) (L is bpy = 2,2‘-bipyridine or phen = 1,10-phenanthroline). The diffusive current obtained from voltammograms at potentials well past E_(1/2) gives an accurate measure of the extent to which the complexes codiffuse with DNA or are free in solution, and this response is not affected by kinetic factors resulting from slow heterogeneous electron transfer. Analysis of the diffusion-limited current using the appropriate binding isotherm provides binding constants in good agreement with those measured by other methods. For the bpy complexes, the ionic strength dependence, the relative binding constants for the 2+ and 3+ forms, and the associated change in E_(1/2) upon DNA binding are in good agreement with the predictions of polyelectrolyte theory where the 3+ ion binds more strongly. For the phen complexes, the reverse trend is observed and is consistent among the absolute binding constants, ionic strength dependence, and E_(1/2) shift; this behavior is ascribed to a hydrophobic interaction. The technique is also applied to two-electron couples based on [(tpy)(L)RuOH_2]^(2+)/[(tpy)(L)RuO]^(2+) that exhibit slow heterogeneous electron transfer; however, these kinetic complications do not prohibit accurate determination of the binding energetics using normal pulse voltammetry. Taken together, the data provide a comprehensive picture of the effects of partial DNA binding on voltammetry, which provides a basis for determining homogeneous kinetic rate constants for electrocatalytic DNA oxidation from voltammograms

Distribution of Metal Complexes Bound to DNA Determined by Normal Pulse Voltammetry

The effects of DNA binding on the normal pulse voltammetry of metal complexes have been investigated. Studies were performed both for oxidation of OsL_3^(2+/3+) and for reduction of CoL_3^(3+/2+) (L is bpy = 2,2‘-bipyridine or phen = 1,10-phenanthroline). The diffusive current obtained from voltammograms at potentials well past E_(1/2) gives an accurate measure of the extent to which the complexes codiffuse with DNA or are free in solution, and this response is not affected by kinetic factors resulting from slow heterogeneous electron transfer. Analysis of the diffusion-limited current using the appropriate binding isotherm provides binding constants in good agreement with those measured by other methods. For the bpy complexes, the ionic strength dependence, the relative binding constants for the 2+ and 3+ forms, and the associated change in E_(1/2) upon DNA binding are in good agreement with the predictions of polyelectrolyte theory where the 3+ ion binds more strongly. For the phen complexes, the reverse trend is observed and is consistent among the absolute binding constants, ionic strength dependence, and E_(1/2) shift; this behavior is ascribed to a hydrophobic interaction. The technique is also applied to two-electron couples based on [(tpy)(L)RuOH_2]^(2+)/[(tpy)(L)RuO]^(2+) that exhibit slow heterogeneous electron transfer; however, these kinetic complications do not prohibit accurate determination of the binding energetics using normal pulse voltammetry. Taken together, the data provide a comprehensive picture of the effects of partial DNA binding on voltammetry, which provides a basis for determining homogeneous kinetic rate constants for electrocatalytic DNA oxidation from voltammograms