Chemical synthesis of tridecanucleoside dodecaphosphate sequence of SV40 DNA

The preparation, by the phosphotriester approach, of d[C-T-A-T-T-C-C-A-G-A-A-G-T] from one tetranucleoside triphosphate and three trinucleoside diphosphate blocks is described. The use of the o-dibromomethylbenzoyl (DBMB) protecting group in oligodeoxyribonucleotide synthesis is described for the first time. Internucleotide linkages are protected by o-chlorophenyl groups which are finally removed by treatment with the N1, N1, N3, N3-tetramethylguanidinium salt of syn-4-nitrobenzaldoxime. The first phosphorylation step (leading to phosphodiester intermediates) is carried out by treatment with o-chlorophenyl phosphorodi-(1,2,4-triazolide) followed by treatment with water and triethylamine. 1-Mesitylenesulphonyl-3-nitro-1,2,4-triazole (MSNT) is used as the activating agent in the second phosphorylation step in which 5'-protected mono- and di-nucleotides are condensed with nucleoside building blocks containing unprotected 3'-hydroxy functions.     

Structure of a T4 hairpin loop on a Z-DNA stem and comparison with A-RNA and B-DNA loops

The synthetic DNA oligomer C-G-C-G-C-G-T-T-T-T-C-G-C-G-C-G crystallizes as a Z-DNA hexamer, capped at one end by a T4 loop. The crystals are monoclinic, space group C2, with a = 57.18 A, b = 21.63 A, c = 36.40 A, beta = 95.22 degrees, and one hairpin molecule per asymmetric unit. The structure of the z-hexamer stem was determined by molecular replacement, and the T4 loop was positioned by difference map methods. The final R factor at 2.1 A resolution for hairpin plus 70 water molecules is 20% for 2 sigma data, with a root-mean-square error of 0.26 A. The (C-G)3 stem resembles the free Z-DNA hexamer with minor crystal packing effects. The T4 loop differs from that observed on a B-DNA stem in solution, or in longer loops in tRNA, in that it shows intraloop and intermolecular interactions rather than base stacking on the final base-pair of the stem. Bases T7, T8 and T9 stack with one another and with the sugar of T7. Two T10 bases from different molecules stack between the C1-G12 terminal base-pairs of a third and fourth molecule, to simulate a T.T "base-pair". Distances between thymine N and O atoms suggest that the two thymine bases are hydrogen bonded, and a keto-enol tautomer pair is favored over disordered keto-keto wobble pairs. The hairpin molecules pack in the crystal in herringbone columns in a manner that accounts well for the observed relative crystal growth rates in a, b and c directions. Hydration seems to be most extensive around the phosphate groups, with lesser hydration within the grooves.     

Direct estimation of base-pair exchange kinetics in oligo-DNA by a combination of NOESY and ROESY experiments.

A new method for the determination of the kinetics of exchange of the imino protons of DNA duplex is reported using a combination NOESY and ROESY experiments at short mixing times (< or = 20 ms). These results have been compared with the commonly used longitudinal relaxation approach through the T1 measurement. To calculate kex and pi ex by ROESY-NOESY experiment, the volume of the cross-peaks between imino protons and water in the NOESY and ROESY spectra have been measured separately from the magnetization term. This work shows that the present approach for the measurement of the kinetics of slow exchanging imino protons of DNA duplex is comparable to the saturation recovery experiment in which the exchange rate can be accelerated by the addition of a base catalyst. The present ROESY-NOESY approach has been found to be particularly useful and reasonably accurate for the measurement of exchange kinetics of both the fast- and slow-exchanging imino protons in DNA duplex both under non-physiological and physiological condition where the saturation recovery method can not be used.     

9-beta-D-Arabinofuranosyl-8-n-butylaminoadenine, a C-8 substituted nucleoside in the anti conformation. Crystallographic and NMR studies

The protein NMR spectrum of 9-beta-D-arabinofuranosyl-8-n-butylaminoadenine shows an unusually low-field 5'-hydroxyl proton resonance, which has been interpreted in terms of an anti glycosidic conformation together with an 05' ... N8 intramolecular hydrogen bond. Confirmatory evidence for this was obtained by an X-ray crystallographic study; in the crystal, the glycosidic angle chi is 52.7 degrees and the sugar pucker is C3' endo-C4' exo.     

Arenesulfonylethoxycarbonyl- a Set of Amino Protecting Groups for DNA and RNA Synthesis

Abstract

Phenyl-, 4-chlorophenyl- and 4-nitrophenylsufonylethoxycarbonyl groups have been reported for the first time as the exocyclic amino protecting groups in nucleoside chemistry. They are all stable under the standard conditions of manipulations in phosphotriester and phosphiteamidite chemistry, they are removable both under the alkaline hydrolytic conditions and also under the influence of non-nucleophilic tertiary bases. N³-Phenyl- and 4-toluenesulfonylethoxycarbonyl derivatives of uridine have been also prepared and characterized by ¹⁵N-NMR spectroscopy, their stabilities under different conditions have been tested.     

Elucidation of the Origin of NOEs And ROEs that Show the Hydration in the Minor and Major Grooves of DNA Duplex with ATTAAT Tract by a Combination of NOESY and ROESY Experiments

Abstract

A combination of NOESY and ROESY experiments (using ammonia as a catalyst across the pH range of 5 to 8.6) has given us a clear understanding regarding the origin of nOes that are attributed to the stereochemical location and the residence time of water in the major and the minor grooves of d⁵'(¹C²C³A⁴T⁵T⁶A⁷A⁸T⁹G¹⁰G)₂ ³' duplex Our conclusions are the following: (i) In the major groove, the presence of ammonia in the buffer does not influence on the process of exchange between bound and bulk water, (ii) It has been found that the observation of the bound water in the minor groove is a result of straight dipole-dipole effect at the physiological pH. (iii) The residence time of water near H2 of adenine (H2A) in the minor groove has been estimated to be in the range of 0.3–0.5ns, which is closer to the residence time of the bound water found on the surface of protein, (iv) The hydration pattern in the minor groove in the physiological pH, under our NMR measurement condition, is similar to the ones found in the X-ray structure, (v) It has been shown that at pH > 8.0 the nOe/rOe intensities of the water-H2A crosspeaks dramatically increase due to dipole-dipole and/or relayed magnetization transfer from H2A to water through ammonia catalyst.     

Improved Model of a LexA Repressor Dimer Bound to recA Operator

Abstract

A complete three dimensional model for the LexA repressor dimer bound to the recA operator site consistent with relevant biochemical and biophysical data for the repressor was proposed from our laboratory when no crystal structure of LexA was available. Subsequently, the crystal structures of four LexA mutants Δ_1–67 S119A, S119A, G85D and Δ_1-67 quadruple mutant in the absence of operator were reported. It is examined in this paper to what extent our previous model was correct and how, using the crystal structure of the operator-free LexA dimer we can predict an improved model of LexA dimer bound to recA operator. In our improved model, the C-domain dimerization observed repeatedly in the mutant operator-free crystals is retained but the relative orientation between the two domains within a LexA molecule changes. The crystal structure of wild type LexA with or without the recA operator cannot be solved as it autocleaves itself. We argue that the ‘cleavable’ cleavage site region found in the crystal structures is actually the more relevant form of the region in wildtype LexA since it agrees with the value of the pre-exponential Arrhenius factor for its auto- cleavage, absence of various types of trans-cleavages, difficulty in modifying the catalytic serine by diisopropyl flourophosphate and lack of cleavage at Arg 81 by trypsin; hence the concept of a ‘conformational switch’ inferred from the crystal structures is meaningless.     

Determination of the Tautomeric Populations of the Y-Base

Abstract

Determination of tautomeric population of the Y-base in 3 by ¹³C-NMR spectroscopy has shown that its most thermodynamically preferred tautomeric form is the N¹-H tautomer (～ 95 %) which explains its chemical reactivity in glycosylation reaction and also in the facile, lewis acid promoted isomerization of wyosine-2′,3′,5-O-triacetate 5 to its N¹-isomer 4. It is likely that the consequence of occurrence of natural wyosine in the thermodynamically unfavoured N³-form is its unusual lability across the glycosyl bond under mild acidic conditions.     

Studies on physiology,zoospore morphology and entomopathogenic potential of the aquatic oomycete: Lagenidium giganteum

The oomycete Lagenidium giganteum, a facultative parasite of mosquito larvae requires exogenous sterols for the genesis of zoospores when grown saprobically. Growth media prepared from oil rich materials such as soy or sunflower seed were very effective inducers of virulent zoospores. The external morphology of zoospores of L. giganteum was studied with the aid of philips scanning electron microscope 515. Zoospores were ovoid, bluntly pointed with the groove parallel to the long axis and 0.7 × 1.4 μm. Insect cell walls are known to contain lipid and chitin. L. giganteum was tested for chitinase activity and found to possess 0.76 ± SD0.14 chitinase activity. Use of oil seed for growth of the organism confirms phospholipase activity. Phospholipase production was studied further by egg-yolk plate method. Presence of these two key enzymes that can initiate host cell damage suggests the entomopathogenic potential of L. giganteum. L. giganteum failed to grow at 37 °C limiting its effectiveness in warmer climates. Introduction of this organism to variety of habitats with various mosquito species will demonstrate the efficacy of the organism as a bioinsecticide. This revised version was published online in June 2006 with corrections to the Cover Date.