Secondary structure prediction and in vitro accessibility of mRNA as tools in the selection of target sites for ribozymes.

We have investigated the relative merits of two commonly used methods for target site selection for ribozymes: secondary structure prediction (MFold program) and in vitro accessibility assays. A total of eight methylated ribozymes with DNA arms were synthesized and analyzed in a transient co-transfection assay in HeLa cells. Residual expression levels ranging from 23 to 72% were obtained with anti-PSKH1 ribozymes compared to cells transfected with an irrelevant control ribozyme. Ribozyme efficacy depended on both ribozyme concentration and the steady state expression levels of the target mRNA. Allylated ribozymes against a subset of the target sites generally displayed poorer efficacy than their methylated counterparts. This effect appeared to be influenced by in vivo accessibility of the target site. Ribozymes designed on the basis of either selection method displayed a wide range of efficacies with no significant differences in the average activities of the two groups of ribozymes. While in vitro accessibility assays had limited predictive power, there was a significant correlation between certain features of the predicted secondary structure of the target sequence and the efficacy of the corresponding ribozyme. Specifically, ribozyme efficacy appeared to be positively correlated with the presence of short stem regions and helices of low stability within their target sequences. There were no correlations with predicted free energy or loop length.

An acido- and photochromic molecular device that mimics triode action.

The photo-controlled shift of pH titration curves, describing the acidochromic behaviour of a spiropyran switch network, was harnessed for the realisation of a molecular triode. The intricate network can be correctly interpreted with respect to the pH dependence of the main involved species.

A succession of substrate induced conformational changes ensures the amino acid specificity of Thermus thermophilus prolyl-tRNA synthetase: comparison with histidyl-tRNA synthetase.

We describe the recognition by Thermus thermophilus prolyl-tRNA synthetase (ProRSTT) of proline, ATP and prolyl-adenylate and the sequential conformational changes occurring when the substrates bind and the activated intermediate is formed. Proline and ATP binding cause respectively conformational changes in the proline binding loop and motif 2 loop. However formation of the activated intermediate is necessary for the final conformational ordering of a ten residue peptide ("ordering loop") close to the active site which would appear to be essential for functional tRNA 3' end binding. These induced fit conformational changes ensure that the enzyme is highly specific for proline activation and aminoacylation. We also present new structures of apo and AMP bound histidyl-tRNA synthetase (HisRS) from T. thermophilus which we compare to our previous structures of the histidine and histidyl-adenylate bound enzyme. Qualitatively, similar results to those observed with T. thermophilus prolyl-tRNA synthetase are found. However histidine binding is sufficient to induce the co-operative ordering of the topologically equivalent histidine binding loop and ordering loop. These two examples contrast with most other class II aminoacyl-tRNA synthetases whose pocket for the cognate amino acid side-chain is largely preformed. T. thermophilus prolyl-tRNA synthetase appears to be the second class II aminoacyl-tRNA synthetase, after HisRS, to use a positively charged amino acid instead of a divalent cation to catalyse the amino acid activation reaction.

Surfactant Mediated Cationic and Anionic Suspension Polymerization of PEG-Based Resins in Silicon Oil: Beaded SPOCC 1500 and POEPOP 1500.

A novel surfactant has been synthesized for use in cationic and anionic ring-opening suspension polymerization of PEG-based macromonomers in silicon oil. A polymer of acrylate esters containing pentamethyldisiloxane and PEG was prepared by radical polymerization. The surfactant can stabilize an emulsion of PEG-based macromonomers, initiator, and solvent in silicon oil such that polymer beads are obtained by ring-opening polymerization, initiated either by a Lewis acid (cationic ring opening) or potassium tert-butoxide (anionic ring opening). The average bead size could be controlled by varying the stirring rate and the amount of surfactant and solvent. The surfactant does not interfere with the polymerization and can be removed together with residual silicon oil by a simple washing procedure.

Diffusion of reagents in macrobeads.

A simple and effective method of measuring diffusion rates of various acylating reagents in macro beads (in this work diameters greater than 570 microm) of amino-functionalized resins is presented. The extent of diffusion at various points of time was determined by treating beads with a staining reagent ("chloranil", 2,3,5,6-tetrachloro-1,4-benzoquinone) that colored the regions of the beads that had not yet been permeated by reagent (the central part of the beads). The volume of unstained resin (permeated part) was compared with the volume of the stained part, and diffusion rate constants were calculated. Factors influencing diffusion such as reagent concentration, solvent, temperature, resin type, and mechanical effects were investigated. The results showed that diffusion was promoted by increased temperature, good swelling of the resin, small reagents, and high concentrations but not by sonication or mechanical agitation.

Crystallization and preliminary crystallographic analysis of Thermus thermophilus leucyl-tRNA synthetase and its complexes with leucine and a non-hydrolysable leucyl-adenylate analogue.

Leucyl-tRNA synthetase from Thermus thermophilus (LeuRSTT) is the first LeuRS to be crystallized. Two crystal forms of the native enzyme have been obtained using the hanging-drop vapour-diffusion method with ammonium sulfate as a precipitant. Crystals of the first form belong to space group I422 and have unit-cell parameters a = b = 312.4, c = 100.4 A. They diffract anisotropically to 3.5 A resolution in the c-axis direction and to only 6 A resolution in the perpendicular direction. Crystals of the second form, which can be obtained native or with leucine or a leucyl-adenylate analogue bound, belong to space group C222(1) and have unit-cell parameters a = 102. 4, b = 154.1, c = 174.3 A. They diffract to 1.9 A resolution and contain one monomer in the asymmetric unit. Selenomethionated LeuRSTT has been produced and crystals of the second form suitable for MAD analysis have been grown.

The crystal structure of asparaginyl-tRNA synthetase from Thermus thermophilus and its complexes with ATP and asparaginyl-adenylate: the mechanism of discrimination between asparagine and aspartic acid.

The crystal structure of Thermus thermophilus asparaginyl-tRNA synthetase has been solved by multiple isomorphous replacement and refined at 2.6 A resolution. This is the last of the three class IIb aminoacyl-tRNA synthetase structures to be determined. As expected from primary sequence comparisons, there are remarkable similarities between the tertiary structures of asparaginyl-tRNA synthetase and aspartyl-tRNA synthetase, and most of the active site residues are identical except for three key differences. The structure at 2.65 A of asparaginyl-tRNA synthetase complexed with a non-hydrolysable analogue of asparaginyl-adenylate permits a detailed explanation of how these three differences allow each enzyme to discriminate between their respective and very similar amino acid substrates, asparagine and aspartic acid. In addition, a structure of the complex of asparaginyl-tRNA synthetase with ATP shows exactly the same configuration of three divalent cations as previously observed in the seryl-tRNA synthetase-ATP complex, showing that this a general feature of class II synthetases. The structural similarity of asparaginyl- and aspartyl-tRNA synthetases as well as that of both enzymes to the ammonia-dependent asparagine synthetase suggests that these three enzymes have evolved relatively recently from a common ancestor.

Ether, carbonate and urethane deoxynucleoside derivatives as prodrugs.

3'-Deoxythymidine and its 3'-azido derivative, 2',3'-dideoxycytidine, 2',3'-dideoxyinosine and 2',3'-dideoxyadenosine have been acylated to form carbonates and urethanes in chemoselective reactions. The nucleosides have been N- and/or O-alkylated by alpha-chloroethyl or chloromethyl alkyl carbonates to form alpha-alkyloxycarbonyloxyethyl or alkyloxycarbonyloxymethyl derivatives. The products are lipophilic in order to facilitate transport through biological membranes and are designed to be cleaved by esterases with liberation of the bioactive nucleoside. Initial esterase cleavage of the alkylated derivatives produces hemiacetals or -aminals which subsequently dissociate to the active nucleoside.

Solid-phase glycosylation of peptide templates and on-bead MAS-NMR analysis: perspectives for glycopeptide libraries.

The efficient solid-phase glycosylation of amino acid side chains (serine (Ser), threonine (Thr), and tyrosine (Tyr)) in peptides was demonstrated with a variety of glycosyl trichloroacetimidate donors in high yields and purities. A novel photolabile linker, with no chiral centre, was introduced to facilitate analysis by both matrix-assisted laser desorption ionisation time of flight (MALDI-TOF) mass spectrometry and nanoprobe magic angle spinning (MAS) NMR spectroscopy. Product analysis by nanoprobe MAS NMR spectroscopy, LC-MS and MALDI-TOF mass spectrometry of the glycosylation reactions indicated that the reactivity order of the hydroxy side-chain functions of amino acids in peptides on the solid-phase was Tyr>Ser>Thr. The nearly quantitative glycosylation yields and the efficient on-bead product analysis by nanoprobe MAS NMR spectroscopy have made a truly solid-phase approach for the synthesis and analysis of glycopeptide libraries possible.

Physical properties of poly(ethylene glycol) (PEG)-based resins for combinatorial solid phase organic chemistry: a comparison of PEG-cross-linked and PEG-grafted resins.

Three series of poly(ethylene glycol) (PEG)-based polymers were synthesized and characterized with respect to their physical properties. Polyoxyethylene-polyoxypropylene (POEPOP), polyoxyethylene-polyoxetane (SPOCC), and polyoxyethylene-polystyrene (POEPS-3) were synthesized respectively by anion polymerization, cation polymerization, and radical polymerization. Both bulk and suspension modes were used to synthesize the polymers from derivatized PEG monomers (PEG 400, PEG 900, and PEG 1500). The three supports were compared with two commercially available PEG-grafted supports (TentaGel S OH, ArgoGel-OH) and two polystyrene supports (aminomethylated polystyrene [PS-NH2] and macroporous aminomethylated polystyrene [PLAMS]) with respect to their swelling properties, loading, NMR spectral quality, as well as solvent and reagent accessibility. Loadings of 0.3-0.7 mmol/g were obtained for the PEG-based resins. Swelling of the PEG-based resins was determined to be higher than that of the PEG-grafted resins and polystyrene supports. The PEG-based resins gave better resolved high-resolution NMR spectra than the PEG-grafted resins when examined by magic angle spinning nanoprobe (MAS) NMR spectroscopy. Moreover, fluorescence quenching of polymer bound 2-amino-benzoate by protonation with p-toluenesulfonic acid showed moderate to fast diffusion through the polymer depending on the solvent and the polymer matrix.