Ribose Units Research Articles

A vibrational spectroscopic study of the metastable form of associated polyinosinic acid

We have studied by Raman and ir spectroscopy the metastable complex formed by the self-association of polyinosinic acid in aqueous solution. The complex is easily prepared by quickly cooling to ca. 0°C a warm solution of the polyribonucleotide to which a small amount of rubidium salt has been added. Upon heating, this metastable form melts cooperatively near 13°C, well below the dissociation temperature of a stable four-stranded complex, which occurs at 47°C in the same conditions. The presence of several components in the stretching-mode region of the carbonyl groups in the vibrational spectra of the metastable complex suggests that it also has a parallel four-stranded structure. The difference in structure between the two forms is believed to be caused by the presence of fewer metal ions in the central channel of the metastable complex, in agreement with conclusions reached in previous investigations. The Raman spectra further show that the ribose units in the metastable form have a C3′-endo conformation, in contrast with the stable form, for which we have previously suggested a mixed C2′-endo/C3′-endo conformation. © 1996 John Wiley & Sons, Inc.

Synthesis of Oligobipyridine Strands Bearing Nucleoside and Amino Acid Side Chains

The synthesis is reported of derivatives of 6,6'-dimethyl-2,2'-bipyridine in which adenosine, thymidine aid tryptophan substituents are introduced at the 4,4'-positions as amide derivatives. Artificial oligonucleoside strands derived from the corresponding trisbipyridyl ligand , in which three bipyridyl ligands are separated by methyleneoxy bridges, have been prepared. The solubility of the nucleoside-substituted ligands in organic solvents is poor, but acylation of the ribose or deoxyribose units present in the ligands provides a mechanism whereby organic solubility can be improved. These ligands may be used for the preparation of metallo-exoreceptors , with the potential for recognition of biological targets such as nucleic acids. Preliminary metal ion complexation studies have been carried out.

Modulation of inflammatory arthritis by inhibition of poly(ADP ribose) polymerase

Poly(ADPR) polymerase (PARP; EC 2.4.2.30) is a nuclear enzyme, which, when activated by oxygen- and nitrogen-radical-induced DNA strand breaks, transfers ADP ribose units to nuclear proteins and initiates apoptosis by depletion of cellular NAD and ATP pools. The present study investigates whether the oxidative stress-dependent activation of PARP plays a role in the etiopathogenesis of arthritis. The antiarthritic reactivity of the biogenic PARP inhibitor nicotinamide was tested in DBA/1 x B10A(4R) mice suffering from potassium peroxochromate-induced arthritis. Daily doses of 4 mmol/kg of NA suppressed the arthritis by 35% and inhibited the phagocytic generation of reactive oxygen species, which increases sixfold during the development of arthritis. The onset, progression, and remission of arthritis correlated positively to the phorbolester-activated respiratory burst of neutrophils and monocytes, and a dose-dependent inhibition of NADPH oxidase activity was determined with human phagocytes. Our data support the hypothesis that oxidative stress-induced alterations in cellular signal transduction pathways play a pivotal role in the development of arthritis, which can be suppressed by the simultaneous inhibition of poly(ADPR) polymerase and NADPH oxidase.

The presence of galactofuranose and ribose units in asparagine-linked oligosaccharides of the digenetic trypanosomatid Endotrypanum schaudinni

It was found that the digenetic trypanosomatid Endotrypanum schaudinni transferred Man 7GlcNAc 2 in protein N-glycosylation. Endo-β- N-acetylglucosamindase H-sensitive oligosaccharides were identified as Man 7GlcNAc 2, Man 6GlcNAc 2, Rib 1Man 6GlcNAc 2 and/or Gal 1Man 6GlcNAc 2, Man 5GlcNAc containing two galactose or ribose units or one of each residues, Rib 1Man 5GlcNAc 2 and Gal 1Man 5GlcNAc 2. The galactoses were in the furanose configuration. Endo-β- N-acetylglucosaminidase H-resistant glycopeptides that were retained by concanavalin A-Sepharose and eluted with α-methylmannoside were found to contain mannose, galactofuranose and ribose units. The presence of galactofuranoses in N-glycoproteins has been reported previously in several monogenetic trypanosomatids but only in one digenetic parasite ( Trypanosoma cruzi). This and a recent publication on the structure of Blastocrithidia culicis N-linked oligosaccharides are the first reports on the presence of ribose in eukaryotic glycoconjugates.

Oxovanadium(IV) complexation by adenosine 5′-di-and -tri-phosphate and nucleotide building blocks

Complex formation between oxovanadium(IV) and the bioligands adenosine 5′-diphosphate (ADP), adenosine 5′-triphosphate (ATP), adenine, adenosine and 2′-deoxyadenosine 5′-diphosphate (dADP) was investigated in aqueous solution by EPR, electronic absorption spectroscopy, and pH-potentiometry. The reaction scheme implies that, in acidic media, nucleotides chelate the metal ion through unshared oxygen atoms of the polyphosphate chain, whereas vicinal cis-oriented hydroxyls of the ribose moiety are the binding set in alkaline solution. Species with mixed co-ordination were also detected. Dihydroxobridged dimers can be formed by ATP and ADP molecules co-ordinating through both the phosphate and ribose residues; however, formation of dihydroxo-bridged dimers with mixed chelation is the most favoured. Owing to the poor affinity of VO2+ towards N-donors, adenine is not able to bind VO2+ within the whole measurable pH range. In adenosine the ribose unit provides the ligand with pairs of vicinal cis-oriented hydroxyls, the right arrangement to form stable five-membered chelated rings. Lack of a C(2′)OH group means that dADP does not possess a pair of cis hydroxyls and only diphosphate chelation is observed with this ligand.

A vibrational spectroscopic study of the self‐association of polyinosinic acid and polyguanylic acid in aqueous solution

We have studied by Raman and ir spectroscopy the structure of self-associated polyinosinic acid and polyguanylic acid in aqueous solution. The results are consistent with the formation of a four-stranded complex, which melts cooperatively near 60 degrees C in the case of poly(I) in the presence of K+ ions. The conformation of the ribose in both systems is mixed C2'-endo/C3'-endo, giving a structure that is intermediate between the extremes proposed previously from x-ray diffraction studies. Characteristic Raman bands for the C2'-endo ribose conformation in polyribonucleotides are identified. The four-stranded structure of poly(I) appears to be very flexible, with approximately 15% of the tetrameric segments being disrupted and approximately 30% of the ribose units adopting a disordered conformation prior to melting. This disordering process increases to approximately 75% above the melting transition, with the remaining approximately 25% of the ribose units keeping an ordered C2'-endo or C3'-endo conformation.

A novel synthesis of “double headed” nucleosides [formula omitted] “reversed” nucleosides

The “double headed” nucleosides 10, 11 and 13 bearing pyrimidine or purine bases attached on C-1 and C-5 positions of single ribose unit have been synthesized following a new synthetic strategy. The strategy rests on the introduction of the first nucleo base at C-5 position of the ribose unit giving “reversed” nucleoside and the subsequent attachment of the second base at C-1 position of the ribose, by classical glycosylation reaction. This order of the base attachments gave much better yields on the “double headed” nucleosides then previously known C-1 then C-5 order of attachments.

The effects of ionising radiation on frozen aqueous RNA: an electron paramagnetic resonance study

Exposure of frozen aqueous solutions of various RNA samples to 60Co γ-rays at 77 K gives well defined parallel components (gz= 2.077) which are assigned to alkoxyl radicals (RO˙) formed from the ribose units. The formation of these radicals, presumably by electron loss from ROH units followed by very rapid proton transfer to water molecules hydrogen bonded to the –OH group, establishes that electron loss from the sugar moieties is significant. No primary sugar radicals have been detected for DNA irradiated under the same conditions. This has been explained in terms of rapid electron-donation from proximal bases (i.e., positive ‘hole’ transfer to a neighbouring base) which must also occur for RNA. For the latter, however, proton-transfer is able to compete With electron-transfer.The major electron-gain and -loss centres have combined EPR spectra that closely resemble that for DNA. Loss is confined to the purine bases and gain to the pyrimidine bases. Unfortunately, addition of an electron to cytosine, thymine and uracil in these polymeric systems generates remarkably similar EPR doublet spectra, and it is, in our view, extremely difficult to make an accurate estimate of the relative yields of these centres by computer synthesis. In the case of DNA, the one-electron adduct of thymine is converted into the 5,6-dihydrothymine radical (˙TH) on warming to ca. 200 K. i.e., protonation at the C6 site of the pyrimidine ring. Spectra for the analogous 5,6-dihydrouracil radical (˙UH) have been recorded, but these features are completely absent on annealing irradiated frozen aqueous RNA systems. Possible reasons for this contrast are discussed.

A vibrational spectroscopic study of the self‐association of 5′‐GMP in aqueous solution

AbstractThe Raman spectra of highly concentrated solutions of 5′‐GMP at neutral and acid pH were recorded in order to better characterize the structure of the self‐aggregates formed in these solutions and their melting behavior. Vibrational coupling of the CO stretching vibrations in tetrameric units at neutral pH is shown to yield a characteristic pattern of two Raman bands at ca. 1730 and 1680 cm−1 (1708 and 1664 cm−1 in D2O), and an iractive mode at 1678 cm−1 in D2O. From the intensity of the 1730‐cm−1 band, proportional to tetramer concentration, and that at 1485 cm−1, which reflects the stacking of the bases, the thermal stability of the self‐associates formed at neutral pH is shown to be higher for stacked tetramers. At acid pH, the melting of the helical aggregates responsible for the formation of a gel is preceded by the freeing of the hydrogen‐bonded phosphate groups, accompanied by a change of conformation from C3′‐endo to C2′‐endo in some of the associated ribose units. Previous spectroscopic results suggesting the formation of tetramers as an intermediate step in the melting of the gel were not reproduced in this study.

The molecular dynamics of nucleosides and nucleotides

The relaxation behaviour of various nucleosides and nucleotides derived from uridine and cytidine has been investigated by the ‘magnetization modes’ formalism. The time evolution of the magnetization is measured via inversion recovery experiments. The diffusion constants as well as the random fields are fitted to the experiments by means of a least-squares procedure. All nucleosides and nucleotides are found to have similar rotation diffusion constants for their ribose units, whereas the rotation diffusion constants for the base units differ. The difference is attributed to a significant internal-jump mobility of the base units in the uridine derivatives. To corroborate the fitted values a cross-check with other relaxation-time measurements has been employed.

Structural Determination of a Cyclic Metabolite of NAD+ with Intracellular Ca2+-mobilizing Activity

Incubation of NAD+ with extracts from sea urchin eggs resulted in production of a metabolite which could mobilize intracellular Ca2+ stores of the eggs. In this study we present structural evidence indicating that the metabolite is a cyclized ADP-ribose having an N-glycosyl linkage between the anomeric carbon of the terminal ribose unit and the N6-amino group of the adenine moiety. In view of this structure we propose cyclic ADP-ribose as the common name for the metabolite. The purification procedure for the metabolite consisted of deproteinizing the incubated egg extracts and sequentially chromatographing the extracts through three different high pressure liquid chromatography (HPLC) columns. The homogeneity of the purified metabolite was further verified by HPLC on a Partisil 5 SAX column. Using radioactive precursor NAD+ with label at various positions it was demonstrated that the metabolite was indeed derived from NAD+ and that the adenine ring as well as the adenylate alpha-phosphate were retained in the metabolite whereas the nicotinamide group was removed. This was confirmed by 1H NMR and two-dimensional COSY experiments, which also allowed the identification of all 12 protons on the two ribosyl units as well as the two protons on the adenine ring. From the chemical shifts of the two anomeric protons it was concluded that the C-1 carbons of both ribosyl units were still bonded to nitrogen. The positive and negative ion fast atom bombardment mass spectra showed (M + Na)+, (M - H + 2Na)+, (M - H)-, and (M - 2H + Na)- peaks at m/z 564, 586, 540, and 562, respectively. Exact mass measurements indicated a molecular weight of 540.0526 for (M - H)-. This together with the constraints imposed by the results from NMR, radioactive labeling, and total phosphate determination uniquely specified a molecular composition of C15H21N5O13P2. Analysis by 1H NMR and mass spectroscopy of the only major breakdown product of the metabolite after prolonged incubation at room temperature established that it was ADP-ribose, thus providing strong support for the cyclic structure.

Orientation of spin-labeled nucleotides bound to myosin in glycerinated muscle fibers

Electron paramagnetic resonance (EPR) spectroscopy of paramagnetic derivatives of ATP has been used to probe the angular distribution of myosin in glycerinated muscle fibers. Three nucleotide spin labels have been prepared with the nitroxide free radical moiety attached, via an ester linkage to either: the 2' or 3' positions of the ribose unit of ATP (SL-ATP), the 2' position of 3' deoxy ATP (2'SL-dATP), or the 3' position of 2' deoxy ATP (3'SL-dATP). In muscle fibers, these nucleotides are quickly hydrolyzed to their diphosphate forms. All three diphosphate analogues bind to the nucleotide site of myosin with similar affinities: rabbit psoas fibers, 7 X 10(3)/M; insect flight muscle, 5 X 10(3)/M; and rabbit soleus muscle, 2 X 10(4)/M. Analysis of the spectra showed that the principal z-axis of the nitroxide attached to bound nucleotides was oriented with respect to the filament axis. The principal axes of 3'SL-dADP and 2'SL-dADP appeared to be preferentially aligned at mean angles of 67 degrees +/- 4 degrees and 55 degrees +/- 5 degrees, respectively. The distribution of probes about these angles can be described by Gaussians with widths of 16 degrees +/- 4 degrees and 13 degrees +/- 5 degrees, respectively. The spectrum of bound SL-ADP was a linear combination of the spectra of the two deoxy analogues. These orientations were the same in the three muscle types examined, indicating a high degree of homology in the nucleotide binding site. Applying static strains as high as 0.2 N/mm2 to muscle fibers caused no change in the orientation of myosin-bound, spin-labeled nucleotides. When muscle fibers were stretched to decrease actin and myosin filament overlap, bound SL-ADP produced EPR spectra indicative of probes with a highly disordered angular distribution. Sodium vanadate and SL-ATP caused fiber stiffness to decrease, and the EPR spectrum of the bound analogue indicated an increase in the fraction of disoriented probes with a concomitant decrease in the fraction of oriented probes. These findings indicate that when myosin is bound to actin its nucleotide site is highly oriented relative to the fiber axis, and when this interaction is removed the orientation of the nucleotide site becomes highly disordered.

Preparation, infrared spectra, and crystal structure of two modifications of the (thymidinato-N3)methylmercury(II) complex

Reaction of CH3HgOH with thymidine (HT) yielded the neutral CH3HgT complex crystallizing as a hydrated or an anhydrous material, depending on preparation conditions. Both forms were examined by X-ray diffraction. The anhydrous variety is monoclinic, space group P21, a = 4.798(6), b = 14.270(8), c = 10.390(4) Å, β = 102.74(9)°, and Z = 2 molecules per cell. The structure was refined on 1552 nonzero MoKα reflections to a conventional R factor of 0.034. The hydrated form belongs to the orthorhombic space group P212121, a = 10.484(3), b = 14.633(3), c = 18.538(5), Z = 8. The structure was refined on 1816 nonzero MoKα reflections to R = 0.036. In both forms, the CH3Hg+ ion is linearly bonded to the deprotonated N(3) site of thymidine. The water molecules and hydroxyl groups in the ribose unit participate in a hydrogen bonding network, in which the carbonyl groups are involved as acceptors. The infrared spectra of the two forms differ significantly only by the absorptions due to the water molecules. By comparing with the spectrum of thymidine, diagnostic regions for complexation with deprotonated thymidine have been proposed

15N-labeled Escherichia coli tRNAfMet, tRNAGlu, tRNATyr, and tRNAPhe. Double resonance and two-dimensional NMR of N1-labeled pseudouridine.

The N1 imino units in Escherichia coli tRNAfMet, tRNAGlu, tRNAPhe, and tRNATyr were studied by 1H-15N NMR using three different techniques to suppress signals of protons not attached to 15N. Two of the procedures, Fourier internuclear difference spectroscopy and two-dimensional forbidden echo spectroscopy permitted 1H and 15N chemical shifts to be measured simultaneously at 1H sensitivity. The tRNAs were labeled by fermentation of the uracil auxotroph S phi 187 on a minimal medium containing [1-15N]uracil. 1H and 15N resonances were detected for all of the N1 psi imino units except psi 13 at the end of the dihydrouridine stem in tRNAGlu. Chemical shifts for imino units in the tRNAs were compared with "intrinsic" values in model systems. The comparisons show that the A X psi pairs at the base of the anticodon stem in E. coli tRNAPhe and tRNATyr have psi in an anti conformation. The N1 protons of psi in other locations, including psi 32 in the anticodon loop of tRNAPhe, form internal hydrogen bonds to bridging water molecules or 2'-hydroxyl groups in nearby ribose units. These interactions permit psi to stabilize the tertiary structure of a tRNA beyond what is provided by the U it replaces.

Induction of Bacterial Differentiation by Adenine- and Adenosine-Analogs and Inhibitors of Nucleic Acid Synthesis

Abstract Several adenine- or adenosine-analogs, which inhibited growth and decreased the intracellular GTP pool, induced sporulation of Bacillus subtilis. The inducers were added to cultures growing in a medium containing excess ammonium ions, glucose, and phosphate in which cells normally cannot differentiate. They included compounds that are modified in the ribose unit (decoyinine, psicofuranine, cordycepin) or are substituted within the purine ring or at the 6-N position of adenosine (6-methylaminopurine, zeatin, 6-anilinopurine, formycin). Their effects on the cellular concentration of nucleotides were also measured. All sporulation inducers except formycin-A caused a decrease of GMP, GDP and GTP, some by inhibiting IMP dehydrogenase and others by inhibiting GMP synthetase. In contrast, formycin-A caused an increase of GMP while GDP and GTP decreased. Therefore, the compound (signal) controlling sporulation is GDP or GTP but not GMP. Antibiotics inhibiting growth by direct inhibition of nucleic acid s...

Effect of aminonucleoside on transcription, methylation, and maturation of ribosomal RNA in SV40-transformed human lung fibroblasts.

The effects of puromycin aminonucleoside (AMS) have been studied in the nucleoli of SV40-transformed human lung fibroblasts with attention to the relationship between transcription, methylation, and maturation of ribosomal RNA (rRNA). Inhibition of the transcription of pre-rRNA became evident between 1 and 2 hr of exposure to AMS, and the degree of inhibition remained approximately constant throughout the remainder of the 18-hr period of study. Methylation of the ribose units of pre-rRNA was inhibited by AMS approximately as much as RNA transcription, but later, suggesting that the inhibition of methylation is a consequence of the lowered rate of RNA transcription. It was also noted that ribose methylation of nucleolar RNA occurs in the absence of concurrent RNA transcription, indicating that rRNA may be methylated post-transcriptionally. Exposure of the fibroblasts to fresh serum had the capacity to increase the rate of nucleolar RNA transcription if the fibroblasts were treated with AMS for periods of less than 2 hr but was ineffective thereafter, confirming that the inhibitory effect of AMS on pre-rRNA transcription is established in approximately 2 hr. On the other hand, processing of the primary transcript (45 S RNA) to the mature rRNA species (28 and 18 S) was inhibited more gradually, with a complete inhibition of rRNA maturation being noted after 18 hr of AMS treatment. These data are consistent with the view that the primary effect of AMS is on the rate of rRNA transcription, with a later effect on its maturation, while RNA methylation is reduced only because of the diminished availability of the RNA substrate.

The low frequency vibrations of the nucleosides: Uridine, cytidine and inosine. Determination of vibrations associated with the ribose ring

The room temperature far i.r. spectra of the nucleoside cytidine and its deuterated analog cytidine- d 3 have been measured from 200 to 100 cm −1. Several spectral features involving hydroxyl deformations have been identified. Between 200 and 400 cm −1 the spectra of the nucleosides cytidine, uridine and inosine have been compared to those of the respective bases cytosine, uracil and hypoxanthine thereby allowing the identification of several bands associated with deformations of the furanose ribose unit. These frequencies are common among the nucleosides investigated and have been compared to calculated frequencies for “ribose” modes in polynucleotides. Upon cooling to 7 K the cytidine and cytidine- d 3 spectra below 200 cm −1 exhibit a remarkable narrowing resembling a “gas-like” spectra, revealing more spectral features than expected from a group theoretical analysis of the unit cell. The low temperature spectra also exhibit an apparent periodicity. Two equally possible explanations are given; one accounts for the observed periodicity via a vibrational exciton-like theory, while the alternative explanation treats the periodicity as accidental.

Structure and serological specificity of the K13-antigenic polysaccharide (K13 antigen) of urinary tract-infective Escherichia coli.

The primary structure of the K13-antigenic polysaccharide (K13 antigen) of Escherichia coli O6:K13:H1 was elucidated by composition, periodate oxidation, Smith degradation, and methylation analysis. The polysaccharide consists of a repeating sequence of 3-linked ribofuranose and 7-linked 3-deoxymannooctulosonic acid (KDO). About 50% of the KDO residues are O-acetylated at position 4 or 5. Measurement of the optical rotary dispersion indicated that in aqueous solution the K13 polysaccharide assumes a secondary structure in which the carboxyl groups of KDO are engaged. The serological specificity of the K13 polysaccharide is expressed through KDO and its O-acetyl substituent, the ribose unit being antigenically silent. There are two populations of anti-K13 antibodies one directed against the charged region of the KDO and the other against the O-acetyl groups.

Quantum-mechanical exploration of the properties of the sugar rings

Theab initio SCF method is used for computing the main electronic properties of the ribose unit of the nucleic acids. The present study is devoted to the ribose in the C3′-endo,gg conformation. The properties investigated include the distribution of the electronic charges, the electrostatic molecular potential around the four oxygens of the unit, the hydration and the Na+ binding schemes studied in the supermolecule approximation. The possibilities of through-water binding of the cation to the sugar are also explored. The predictions of the computation in particular with regard to cation binding to the ribose ring are correlated with recent experimental results.

Separation of oligo(adenosine diphosphate ribose) fractions with various chain lengths and terminal structures

Oligo(adenosine diphosphate ribose) preparations with chain lengths of 3 to 10 adenosine diphosphate ribose units were fractionated according to their chain lengths and their terminal structures by hydroxyapatite column chromatography and then polyacrylamide gel electrophoresis. The peak fractions from the hydroxyapatite column were each separated into two distinct subfractions by gel electrophoresis. The two subfractions were found to differ in chain length and terminal structure. A linear correlation was observed between the mobility and the logarithm of the chain length of oligo(adenosine diphosphate ribose) on gel electrophoresis, irrespective of the terminal structure.