Method Development And Applications To Screening And Characterization Of Rrna-Targeting Small Molecules

A series of single ring aminoglycoside analogues was tested for binding to a model RNA representing the A site using electrospray ionization mass spectrometry (ESI-MS). Several of the synthetic analogues with low molecular weights were found to bind to the RNA with affinities comparable to the parental aminoglycoside neamine, with apparent dissociation constants in the low micromolar range. Salt dependence of the affinity constants for the single ring analogues revealed a predominantly electrostatic binding mode. Footprinting experiments revealed that one of the compounds (DHR23) has a similar binding site as the antibiotic paromomycin. DMS chemical probing results also suggest that the binding of DHR23 to the A site leads to stabilization of the stacked-in conformation of A1492 and A1493. To aid in the ligand identification process, a modified FID assay for screening RNA-binding ligands was established using 3-methyl-2-((1-(3-(trimethylammonio)propyl)-4-quinolinylidene)methyl)benzothiazolium (TO-PRO) as the fluorescent indicator. ESI-MS results provide direct evidence that correlates the reduction in fluorescence intensity observed in the FID assay with displacement of the dye molecule from RNA. The assay was successfully applied to screen a variety of RNA-binding ligands with a set of small hairpin RNAs. Ligands that bind with moderate affinity to the chosen RNA constructs were identified. Furthermore, the specificity of one compound, DHR23 as well as a range of other ligands were tested for binding to a set of RNA models, as well as the modified and unmodified decoding region RNA constructs. The results show that DHR23 has preferred binding to structured RNA as compared to ssRNA, as well as a modest preference for the A-site RNA. Also our results indicate that modified nucleotides at or near the ligand-binding pocket may affect binding affinity of small molecules. In summary, the results from this work have shown that generation of compounds based on these simplified structures in combination with FID screening may lead to selective reagents for RNA internal bulges, loops, mismatches, or other unique secondary structure elements

Assessment of fetal wastage in pigs slaughtered at Kumasi abattoir, Ghana

There had been dearth of information on the level of fetal wastages in slaughtered pigs in West African countries other than Nigeria. This study evaluates the fetal losses due to slaughtering of pregnant pigs at the Kumasi abattoir in Kumasi, Ghana. The ages of the fetuses were estimated using standard technique with the influence of sex, age and breed of pig on fetal loss being evaluated. Data were subjected to descriptive and quantitative analysis. 2817 pigs were slaughtered during the period of the study with a monthly average of 563.1739(61.73%) were sows and 430(29.27%) were pregnant. 141(28.20%) sows were slaughtered in January, constituting the highest throughout the period of the study with corresponding fetal loss of 616(76.60%). Most of the fetuses wasted in this study were recorded in the second and third trimester (83.51%). The percentage of fetal wastage (the total number of fetuses wasted divided by the total number of pigs slaughtered) was 2117(75%) in this study. The results revealed high incidence of fetal losses at the Kumasi abattoir. There is need to advocate for routine veterinary checks and interventions in order to reduce the high level of fetal wastage which call for strategic planning and decision-making on animal food security in Ghana.Keywords: Abattoir, Fetal wastage, Slaughtered pigs, Ghan

Bio-orthogonal Fluorescent Labelling of Biopolymers through Inverse-Electron-Demand Diels–Alder Reactions

Bio-ort hogona llabellin gschemes based on inverse-elec tron- deman dDiels–Ald er (IEDDA) cycloa ddition have attracted much attention in chem ical biology recently .The appeal ing features of this reactio n, such as the fast reactio nkinetics, fully bio-ort hogonal nature and high selectiv ity,have helped chem i- cal biologists gain deeper understandi ng of biochemic al pro- cesses at the molecular level.Listing the compo nents and dis- cussing the possib ilities andlimitations of thesereagent s, we provid earecent snapshot of the field of IEDDA -based biomo- lecular manipulatio nwith special focus on fluores cent modula- tion approaches throug hthe use of bio-orthogon alized build- ing blocks. At the end, we discuss challenges that need to be addres sed for further develop ments in order to overcome recent limita tions and to enabl eresearchers to answer biomo - lecular quest ions in more detail

Protected N-Acetyl Muramic Acid Probes Improve Bacterial Peptidoglycan Incorporation via Metabolic Labeling

Metabolic glycan probes have emerged as an excellent tool to investigate vital questions in biology. Recently, methodology to incorporate metabolic bacterial glycan probes into the cell wall of a variety of bacterial species has been developed. In order to improve this method, a scalable synthesis of the peptidoglycan precursors is developed here, allowing for access to essential peptidoglycan immunological fragments and cell wall building blocks. The question was asked if masking polar groups of the glycan probe would increase overall incorporation, a common strategy exploited in mammalian glycobiology. Here, we show, through cellular assays, that E. coli do not utilize peracetylated peptidoglycan substrates but do employ methyl esters. The 10-fold improvement of probe utilization indicates that (i) masking the carboxylic acid is favorable for transport and (ii) bacterial esterases are capable of removing the methyl ester for use in peptidoglycan biosynthesis. This investigation advances bacterial cell wall biology, offering a prescription on how to best deliver and utilize bacterial metabolic glycan probes.Fil: Brown, Ashley R.. University of Delaware; Estados UnidosFil: Wodzanowski, Kimberly A.. University of Delaware; Estados UnidosFil: Santiago, Cintia Cecilia. University of Delaware; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hyland, Stephen N.. University of Delaware; Estados UnidosFil: Follmar, Julianna L.. University of Delaware; Estados UnidosFil: Asare Okai, Papanii. University of Delaware; Estados UnidosFil: Grimes, Catherine Leimkuhler. University of Delaware; Estados Unido

Cu(II)-Based Paramagnetic Probe to Study RNA–Protein Interactions by NMR

Paramagnetic NMR techniques allow for studying three-dimensional structures of RNA–protein complexes. In particular, paramagnetic relaxation enhancement (PRE) data can provide valuable information about long-range distances between different structural components. For PRE NMR experiments, oligonucleotides are typically spin-labeled using nitroxide reagents. The current work describes an alternative approach involving a Cu­(II) cyclen-based probe that can be covalently attached to an RNA strand in the vicinity of the protein’s binding site using “click” chemistry. The approach has been applied to study binding of HIV-1 nucleocapsid protein 7 (NCp7) to a model RNA pentanucleotide, 5′-ACGCU-3′. Coordination of the paramagnetic metal to glutamic acid residue of NCp7 reduced flexibility of the probe, thus simplifying interpretation of the PRE data. NMR experiments showed attenuation of signal intensities from protein residues localized in proximity to the paramagnetic probe as the result of RNA–protein interactions. The extent of the attenuation was related to the probe’s proximity allowing us to construct the protein’s contact surface map