RNA and RNA-protein complexes as targets for therapeutic intervention.

Today, the majority of pharmaceuticals developed to treat cancers and viral/bacterial infections target cellular, bacterial or viral proteins known to be associated with a given pathology. Although proteins are the focus of most current drug discovery efforts, exciting new research has recently begun which aims to exploit ribonucleic acid (RNA) and RNP particles as novel targets for pharmaceutical development. These RNA-targeted research efforts have been fueled by an increased appreciation for the central role played by RNA and RNA-protein interactions in many biological processes and diseases, together with a better understanding of RNA structure and an improvement in biophysical/biochemical techniques available to study RNA.

Effect of malonyl malonanilide dimers on the thermal stability of nitrocellulose.

Three malonanilide dimers (MA2-MA4) were prepared by the reaction of ethyl malonate with malonanilide derivatives (M2-M4). These dimers were investigated as new stabilizers for nitrocellulose. The evaluation process has been performed through Bergman-Junk (BJ) test, thermal analysis measurements (TGA and DSC) and kinetic parameters calculations (E(a)).

S(3)E-E.COSY methods for the measurement of (19)F associated scalar and dipolar coupling constants.

A (1)H-(19)F spin state selective excitation (S(3)E) pulse sequence element has been applied in combination with (1)H homonuclear mixing to create E.COSY-type experiments designed to measure scalar J(HF2') and J(HH2') and residual dipolar D(HF2') and D(HH2') couplings in 2'-deoxy-2'-fluoro-sugars. The (1)H-(19)F S(3)E pulse sequence element, which resembles a simple INEPT sequence, achieves spin-state-selective correlation between geminal (1)H-(19)F spin pairs by linear combination of in-phase (19)F magnetization and anti-phase magnetization evolved from (1)H.

Measurement and application of 1H-19F dipolar couplings in the structure determination of 2'-fluorolabeled RNA.

Residual dipolar couplings can provide powerful restraints for determination and refinement of the solution structure of macromolecules. The application of these couplings in nucleic acid structure elucidation can have an especially dramatic impact, since they provide long-range restraints, typically absent in NOE and J-coupling measurements. Here we describe sensitive X-filtered-E.

Association of an RNA kissing complex analyzed using 2-aminopurine fluorescence.

The fluorescent probe, 2-aminopurine-2'-O-methyl riboside (2-AP) has been selectively incorporated at adenosine positions in stem-loops (so called R1inv and R2inv), derived from the ColE1 plasmid encoded RNA I and RNA II transcripts, that interact to form stable loop-loop kissing complexes and bind the RNA one modulator (Rom) protein, such that fluorescence-detected stopped-flow and equilibrium methods could be used to study the detailed mechanism of this RNA-RNA interaction. Formation of loop-loop kissing complexes between R1inv and R2inv hairpins, substituted with 2-AP at positions in the complementary loops, results in a 5-10-fold fluorescence emission decrease (F(max) = 370 nm), which provides a sensitive measure for the binding reaction. The 2-AP substituted complexes are found to have equilibrium binding properties (average K(D) = 2.

Stressing-out DNA? The contribution of serine-phosphodiester interactions in catalysis by uracil DNA glycosylase.

The DNA repair enzyme uracil DNA glycosylase (UDG) pinches the phosphodiester backbone of damaged DNA using the hydroxyl side chains of a conserved trio of serine residues, resulting in flipping of the deoxyuridine from the DNA helix into the enzyme active site. We have investigated the energetic role of these serine-phosphodiester interactions using the complementary approaches of crystallography, directed mutagenesis, and stereospecific phosphorothioate substitutions. A new crystal structure of UDG bound to 5'-HO-dUAAp-3' (which lacks the 5' phosphodiester group that interacts with the Ser88 pinching finger) shows that the glycosidic bond of dU has been cleaved, and that the enzyme has undergone the same specific clamping motion that brings key active site groups into position as previously observed in the structures of human UDG bound to large duplex DNA substrates.

Molecular modelling study of HIV p17gag (MA) protein shell utilising data from electron microscopy and X-ray crystallography.

The matrix protein p17gag (MA) is a product of proteolytic cleavage of the gag gene encoded polyprotein (pr55gag) and is formed when HIV particles undergo the process of maturation. The MA protein is associated with the inner surface of the viral membrane and determines the overall shape of the virion. Previous studies have shown the existence of trimers of MA in solution and in the crystalline state.

Mechanism of neomycin and Rev peptide binding to the Rev responsive element of HIV-1 as determined by fluorescence and NMR spectroscopy.

Rev is an essential HIV-1 regulatory protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome and is involved in transport of unspliced or partially spliced viral mRNA from the cell nucleus to the cytoplasm. Previous studies have shown that a short alpha-helical peptide derived from Rev (Rev 34-50), and a truncated form of the RRE sequence provide a useful in vitro system to study this interaction while still preserving the essential aspects of the native complex. We have selectively incorporated the fluorescent probe 2-aminopurine 2'-O-methylriboside (2-AP) into the RRE sequence in nonperturbing positions (A68 and U72) such that the binding of both Rev peptide and aminoglycoside ligands could be characterized directly by fluorescence methods.

Heteronuclear NMR and crystallographic studies of wild-type and H187Q Escherichia coli uracil DNA glycosylase: electrophilic catalysis of uracil expulsion by a neutral histidine 187.

The nature of the putative general acid His187 in the reaction catalyzed by Escherichia coli uracil DNA glycosylase (UDG) was investigated using X-ray crystallography and NMR spectroscopy. The crystal structures of H187Q UDG, and its complex with uracil, have been solved at 1.40 and 1.

Role of electrophilic and general base catalysis in the mechanism of Escherichia coli uracil DNA glycosylase.

Escherichia coli uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil bases in DNA by flipping the deoxyuridine from the DNA helix [Stivers, J. T., et al.

Crystal structure of Escherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: structure and glycosylase mechanism revisited.

The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil residues from single-stranded or duplex DNA, producing free uracil and abasic DNA. Here we report the high-resolution crystal structures of free UDG from Escherichia coli strain B (1.60 A), its complex with uracil (1.

Kinetic mechanism of damage site recognition and uracil flipping by Escherichia coli uracil DNA glycosylase.

The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes hydrolytic cleavage of the N-glycosidic bond of premutagenic uracil residues in DNA by flipping the uracil base from the DNA helix. The mechanism of base flipping and the role this step plays in site-specific DNA binding and catalysis by enzymes are largely unknown. The thermodynamics and kinetics of DNA binding and uracil flipping by UDG have been studied in the absence of glycosidic bond cleavage using substrate analogues containing the 2'-alpha and 2'-beta fluorine isomers of 2'-fluoro-2'-deoxyuridine (Ubeta, Ualpha) positioned adjacent to a fluorescent nucleotide reporter group 2-aminopurine (2-AP).

Feasibility of Applying a Stable Isotopic Tracer for Direct Determination of Dry Particulate Deposition to Soybean Plants.

A stable rare-earth isotopic tracer was used to measure the deposition of KNO particles on soybean leaves by direct measurement of the tracer on the plant surfaces by thermal-ionization mass spectrometry. Submicrometer particles, made from a solution containing 3 |mg mL neodymium isotope (Nd, 87.9%) and 1,000 mg mL KNO, were dispersed with a two-fluid nozzle and released upwind of a soybean field.

A molecular dynamics study of Fe2S2 putidaredoxin: multiple conformations of the C-terminal region.

Putidaredoxin (Pdx) plays an essential role as an electron donor and effector in the biochemical cycle involving cytochrome P450cam. Only recently has an NMR-derived structure for this protein been published, but because of the presence of a paramagnetic Fe2S2 center, the NMR assignment could not be completed for residues within a region of 8 A around the active site. That region was modeled by homology with a related protein.

Estimation of the absorbed dose in radiation-processed food--2. Test of the EPR response function by an exponential fitting analysis.

The use of electron paramagnetic resonance spectroscopy to accurately evaluate the absorbed dose to radiation-processed bones (and thus meats) is examined. Additive re-irradiation of the bone produces a reproducible dose response function which can be used to evaluate the initial dose by back-extrapolation. It was found that an exponential fit (vs linear or polynomial) to the data provides improved accuracy of the estimated dose.

Estimation of the absorbed dose in radiation-processed food--1. Test of the EPR response function by a linear regression analysis.

Free radicals produced in chicken bone tissue by 137Cs gamma-rays were measured using electron paramagnetic resonance spectroscopy. The yield of radicals was found to be proportional to the absorbed dose. Additive re-irradiation of previously irradiated bones is the basis of a method to estimate the absorbed dose in radiation-processed foods.

Ascorbic and dehydroascorbic acids measured in plasma preserved with dithiothreitol or metaphosphoric acid.

We describe a rapid method for accurately and precisely measuring ascorbic acid and dehydroascorbic acid in plasma. Total analysis time is less than 10 min, replicate analyses of a single pool provide precision less than or equal to 2%, and values measured in supplemented samples agree with known concentrations of 4.68 and 11.