Charged nucleobases and their potential for RNA catalysis

Catalysis in living cells is carried out by both proteins and RNA. Protein enzymes have been known for over 200 years, but RNA enzymes, or "ribozymes", were discovered only 30 years ago. Developing insight into RNA enzyme mechanisms is invaluable for better understanding both extant biological catalysis as well as the primitive catalysis envisioned in an early RNA-catalyzed life. Natural ribozymes include large RNAs such as the group I and II introns; small RNAs such as the hepatitis delta virus and the hairpin, hammerhead, VS, and glmS ribozymes; and the RNA portion of the ribosome and spliceosome. RNA enzymes use many of the same catalytic strategies as protein enzymes, but do so with much simpler side chains. Among these strategies are metal ion, general acid-base, and electrostatic catalysis. In this Account, we examine evidence for participation of charged nucleobases in RNA catalysis. Our overall approach is to integrate direct measurements on catalytic RNAs with thermodynamic studies on oligonucleotide model systems. The charged amino acids make critical contributions to the mechanisms of nearly all protein enzymes. Ionized nucleobases should be critical for RNA catalysis as well. Indeed, charged nucleobases have been implicated in RNA catalysis as general acid-bases and oxyanion holes. We provide an overview of ribozyme studies involving nucleobase catalysis and the complications involved in developing these mechanisms. We also consider driving forces for perturbation of the pK(a) values of the bases. Mechanisms for pK(a) values shifting toward neutrality involve electrostatic stabilization and the addition of hydrogen bonding. Both mechanisms couple protonation with RNA folding, which we treat with a thermodynamic formalism and conceptual models. Furthermore, ribozyme reaction mechanisms can be multichannel, which demonstrates the versatility of ribozymes but makes analysis of experimental data challenging. We examine advances in measuring and analyzing perturbed pK(a) values in RNA. Raman crystallography and fluorescence spectroscopy have been especially important for pK(a) measurement. These methods reveal pK(a) values for the nucleobases A or C equal to or greater than neutrality, conferring potential histidine- and lysine/arginine-like behavior on them. Structural support for ionization of the nucleobases also exists: an analysis of RNA structures in the databases conducted herein suggests that charging of the bases is neither especially uncommon nor difficult to achieve under cellular conditions. Our major conclusions are that cationic and anionic charge states of the nucleobases occur in RNA enzymes and that these states make important catalytic contributions to ribozyme activity. We conclude by considering outstanding questions and possible experimental and theoretical approaches for further advances.     

Stable dye-labelled oligonucleotide-nanoparticle conjugates for nucleic acid detection

Metallic nanoparticles functionalized with oligonucleotides are used for a number of nucleic acid detection strategies. However, oligonucleotide-nanoparticle conjugates suffer from a lack of stability when exposed to certain conditions associated with DNA detection assays. In this study, we report the synthesis of thiol and thioctic acid-modified oligonucleotide gold nanoparticle (OGNs) conjugates functionalized with a dye label and varying spacer groups. The thioctic acid-modified conjugates exhibit increased stability when treated with dithiothreitol (DTT) compared to the more commonly used thiol modification. When the dye labelled oligonucleotide nanoparticle conjugates are exposed to the same conditions there is a pronounced increase in the stability for both thioctic acid and thiol modified sequences. These results open up the possibility of simply using a dye label to enhance the stability of oligonucleotide-nanoparticle conjugates in DNA detection assays where the enhanced stability of the conjugate system can be advantageous in more complex biological environments.     

Mining the cinnabaramide biosynthetic pathway to generate novel proteasome inhibitors

The cinnabaramides and salinosporamides are mixed PKS/NRPS natural products isolated from a terrestrial streptomycete and a marine actinomycete, respectively. They interfere with the proteasome and thus potentially inhibit the growth of cancer cells. The compounds exhibit a γ-lactam-β-lactone bicyclic ring structure attached to a cyclohexenyl unit and a PKS side chain. As a first step towards improving anticancer activity and permitting genetic approaches to novel analogues, we have cloned and characterized the cinnabaramide biosynthetic genes from Streptomyces sp. JS360. In addition to the expected PKS and NRPS genes, the cluster encodes functionalities for the assembly of the hexyl side chain precursor. The corresponding enzymes exhibit relaxed substrate specificities towards a number of synthesized precursors, enabling production of novel chlorinated cinnabaramides. These were isolated and analyzed for activity, revealing that derivatives bearing a chlorine atom in the PKS side chain show higher inhibitory potentials towards the proteasome's proteolytic subunits (especially the trypsin and chymotrypsin units) and higher cytotoxicities towards human tumor cell lines than the parent cinnabaramide A. Although their activities towards the proteasome were weaker than that of salinosporamide A, the cinnabaramides were found to inhibit the growth of various fungi with greater potency.     

Looking to the future: biomarkers in the management of pancreatic adenocarcinoma

The incidence and mortality of pancreas cancer converge. There has been little advancement in the treatment of pancreas cancer since the acceptance of gemcitabine as the standard therapy. Unfortunately, the efficacy of gemcitabine is dismal. While there is much discussion for the development of biomarkers to help direct therapy in this area, there is little action to move them into clinical practice. Herein, we review potential pancreatic cancer biomarkers and discuss the limitations in their implementation.     

The critical role of water in spider silk and its consequence for protein mechanics

Due to its remarkable mechanical and biological properties, there is considerable interest in understanding, and replicating, spider silk's stress-processing mechanisms and structure-function relationships. Here, we investigate the role of water in the nanoscale mechanics of the different regions in the spider silk fibre, and their relative contributions to stress processing. We propose that the inner core region, rich in spidroin II, retains water due to its inherent disorder, thereby providing a mechanism to dissipate energy as it breaks a sacrificial amide-water bond and gains order under strain, forming a stronger amide-amide bond. The spidroin I-rich outer core is more ordered under ambient conditions and is inherently stiffer and stronger, yet does not on its own provide high toughness. The markedly different interactions of the two proteins with water, and their distribution across the fibre, produce a stiffness differential and provide a balance between stiffness, strength and toughness under ambient conditions. Under wet conditions, this balance is destroyed as the stiff outer core material reverts to the behaviour of the inner core.     

Optimization of the procedure to burn textile fabrics by cone calorimeter: Part I. Combustion behavior of polyester

The aim of this study was the optimization of cone calorimeter to determine the burning behavior of textile fabrics. In particular, the combustion behavior of commercial polyester textile fabrics with varying densities was studied in terms of time to ignition (TTI), heat release rate (HRR), and relative peak (pkHRR) were monitored. Reproducibility and repeatability of the data have been verified by the influence of instrument variable including incident heat flux, the temperature of ceramic backing pads and retaining grid used during sample mounting as well as sample weight (as the number of fabric layers), the density of textiles, and the relative humidity. A low reproducibility has been obtained when a wire grid was not used to stabilize samples during the tests. The effects of these variables on TTI and pkHRR were observed. Copyright © 2010 John Wiley & Sons, Ltd.     

Isolation of High Oleate Recombinants in Peanut by Near Infra‐Red Spectroscopy and Confirmation With Allele Specific Polymerase Chain Reaction Marker

Near infrared (NIR) spectrophotometer offers rapid, noninvasive, nondestructive, and high‐throughput phenotyping of seed samples for use in agriculture and industry. In this study, a reflectance‐based NIR spectrophotometer was calibrated and used for the isolation of desirable higher‐oleic‐acid peanut recombinants from single‐seed‐derived segregating populations at F₇ and F₈ generations. A calibration model was developed through partial least‐square regression using wet chemistry data from 158 peanut genotypes. Desirable prediction for oil, palmitic acid, oleic acid, and linoleic acid in intact seed was obtained based on this calibration. It detected significant high correlations (r) and coefficient of determination (R²) between the actual gas chromatography values and NIR predicted values of fatty acid profile in another 123 peanut genotypes that were generated from crosses involving a high‐oleate mutant and Spanish bunch varieties with early maturity. From this recombinant single‐seed‐derived progenies, 15 higher‐oleate recombinants were isolated and later genotyped through an in‐house developed polymerase chain reaction‐based allele specific marker. The present study has generated high‐oleate peanut recombinants with early maturity in Spanish bunch background. The breeding materials generated here will be evaluated for yield attributing traits at different locations in future.     

The High‐Pressure Characterization of Energetic Materials: 1,4‐Dimethyl‐5‐Aminotetrazolium 5‐Nitrotetrazolate

In‐situ high‐pressure room temperature synchrotron X‐ray diffraction and infrared microspectroscopy were used to examine the structural and vibrational properties and the equation of state of 1,4‐dimethyl‐5‐aminotetrazolium 5‐nitrotetrazolate (DMATNT). The X‐ray measurements show a smoothly varying pressure‐volume relationship to 20 GPa. However, the anisotropic ratios of the unit cell parameters reveal a discontinuity near 3.3 GPa, which can be attributed to an irreversible isostructural phase transition. A significant increase in the Infrared spectral intensity near this pressure coupled with Dayvdov splitting of the NO₂ bending and scissoring modes suggest the transition results in a skewing of the NO₂ groups and increasing asymmetry of the hydrogen bonding sublattice.     

β-Selective C−H Arylation of Electron-Deficient Thiophenes,Pyrroles, and Furans

A general β-C−H arylation of electron-deficient thiophenes, pyrroles, and furans has been developed using ligand-modulated palladium catalyst. The use of a modified norbornene is crucial for reversing the conventional α-selectivity of these substrates. This method features good yields, high β-selectivity, and good tolerance of functional groups.     

(11<Emphasis Type="Italic">Z</Emphasis>,13<Emphasis Type="Italic">E</Emphasis>)-Hexadecadien-1-yl Acetate: Sex Pheromone of the Grass Webworm <Emphasis Type="Italic">Herpetogramma licarsisalis</Emphasis>—Identification,Synthesis, and Field Bioassays

The grass webworm Herpetogramma licarsisalis (Lepidoptera: Crambidae), which has recently established in pasture in Northland, New Zealand, is an important pest of many tropical and subtropical grasses. Two pheromone components, (Z)-11-hexadecen-1-yl acetate (Z11–16:Ac) and (11Z,13E)-hexadecadien-1-yl acetate (Z11,E13–16:Ac), were identified in pheromone gland extracts of female moths by gas chromatography (GC), GC-electroantennographic detection, and GC-mass spectrometry in conjunction with microchemical tests (dimethyldisulfide and 4-methyl-1,2,4-triazoline-3,5-dione derivatizations). Z11,E13–16:Ac and its geometric isomer (11E,13Z)-hexadecadien-1-yl acetate (E11,Z13–16:Ac) were synthesized via stereoselective Wittig reactions, and the identity of the diene present in the pheromone glands was confirmed to be Z11,E13–16:Ac. Field bioassays at Indooroopilly in Brisbane, Australia, established that Z11,E13–16:Ac was necessary and sufficient for attraction of male grass webworm moths and that the corresponding alcohol, (11Z,13E)-hexadecadien-1-ol (Z11,E13–16:OH), had a strong inhibitory effect on trap catches at the ratios tested. When mixed with Z11,E13–16:Ac in various ratios, Z11–16:Ac had no effect on the attractiveness of lures. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.