Fast atom bombardment combined with tandem mass spectrometry for determining structures of small oligonucleotides

A study of small (n = 3 to 6) oligonucleotide and the metastable and collisionally activated decompositions of their (M-H)- species desorbed by using fast atom bombardment (FAB) is reported. Data were obtained for both ribo- and 2'-deoxyribotrinucleotides and for 2'-deoxyribotetra-, penta-, and hexanucleotides. The favored metastable decompositions of all of the oligonucleotides studied are eliminations of neutral CONH and loss of BH, where B is the base moiety. The BH elimination, however, provides little sequence information in the higher oligonucleotides and the process is more indicative of the different bases present in the oligomer. The chemistry observed upon collisional activation changes as one goes from trinucleotides to hexanucleotides. The formation of sequence ions is more facile for processes involving the 3' terminus, allowing the sequence to be determined. As one goes to the higher oligonucleotides, however, several different competitive fragmentation processes become as facile as or more facile than the reactions giving the sequence ions. This hinders proper ion assignments and makes sequence determination difficult.     

Structural requirements of (2'-5') oligoadenylate for protein synthesis inhibition in human fibroblasts.

The structural requirements of (2'-5')-oligoadenylic acid (pppA(2'p5'A)x, X greater than or equal to 1 or (2'-5'An) for inhibition of protein synthesis in cells were examined with a modified calcium-coprecipitation technique, using a series of trinucleotide analogs (pppA2'p5'A2'p5'N, N=rC, rG, rU, T, dC, dG, dA). In this system both the degree and the duration of the inhibition of protein synthesis were dependent on the added concentration of (2'-5')A3. Of all the heterotrimers, only the deoxy A derivative was active as an inhibitor of protein synthesis, while the other members of the analog series were found to have no inhibitory effects. In competition experiments between (2'-5')A3 and the non-active analogs, three heterotrimers were shown to reduce the activity of (2'-5')A3 in protein inhibition. In contrast, the dephosphorylated (2'-5')A3 had no inhibitory effect and was not effective in blocking (2'-5')A3. These results indicate that the 5'-terminal triphosphate is important for binding of (2'-5')A3 to the site of (2'-5')An action and the adenine base at the 2'-terminus is important for activating the machinery responsible for protein synthesis inhibition in the cells, most likely the (2'-5')An-activated nuclease.     

Fast atom bombardment combined with tandem mass spectrometry for the determination of nucleosides

The positive and negative ion fast atom bombardment (FAB) mass spectra and fast atom bombardment collisionally activated decomposition (CAD) spectra of a series of nucleosides and two dinucleotides are reported. The nucleosides studied are substituted forms of guanosine, adenosine, nebularine, tubercidin, uridine, and related pyrimidines. The FAB and CAD data both contain similar information. The CAD spectra are found to provide some structural information not found in the FAB mass spectra. Tandem mass spectrometry also allows emphasis to be put on weak fragments which are either not observed in the FAB mass spectrum or are lost in the matrix ion signals.     

Synthesis and RNAse L binding and activation of a 2-5A-(5')-DNA-(3')-PNA chimera, a novel potential antisense molecule

Fully automated solid-phase synthesis gave access to a hybrid in which 5'-phosphorylated-2'-5'-linked oligoadenylate (2-5A) is connected to the 5'-terminus of DNA which, in turn, is linked at the 3'-end to PNA [2-5A-(5')-DNA-(3')-PNA chimera]. This novel antisense molecule retains full RNase L activation potency while suffering only a slight reduction in binding affinity.     

Biosynthesis of biopterin. Studies on the mechanism of 6-pyruvoyltetrahydropteridine synthase

[1'-3H]- and [2'-3H]dihydroneopterin triphosphate (NH2TP) were prepared enzymatically from [4-3H]- and [5-3H]glucose and converted to tetrahydrobiopterin (BH4) by an extract from bovine adrenal medulla. The formation of BH4 from both [1'-3H]- and [2'-3H]-NH2TP proceeds with virtually complete loss of the respective tritium label. The breaking of the CH-bond at C-1' is characterized by a kinetic isotope effect of 2.6 +/- 0.5. A smaller kinetic isotope effect of 1.5 +/- 0.2 was found for the breaking of the CH-bond at C-2'.     

Resistance towards exonucleases of dinucleotides with stereochemically altered internucleotide phosphate bonds

Kinetic constants for the hydrolytic susceptibility of the internucleotide phosphate bond in normal dinucleotides [e.g., 2'-deoxycytidylyl-(3'>5')-2'-deoxyuridine (dCpdU) and 2'-deoxyadenylyl-(3'-->5')-2'-deoxycytidine (dApdC)] and isomeric dinucleotides [e.g., 2'-deoxycytidylyl-(3'-->5')-1'-deoxy-2'-isouridine (dCpisodU) and 1'-deoxy-2'-isoadenylyl-(3'-->5')-2'-deoxycytidine (isodApdC)], toward 5'- and 3'-exonucleases, phosphodiesterase I (PDE I) and phosphodiesterase II (PDE II) were experimentally determined and remarkable differences emerged. The study is of importance in the discovery of nuclease-stable inhibitors of HIV integrase, but may also have ramifications in the area of anti-sense oligonucleotides of therapeutic interest.     

Studies of oligoadenylate formation on a poly(U) template

We have studied a variety of condensation reactions involving poly (U) as template and isomeric adenosine dinucleotides as substrates. We find that [3'-5']-linked dinucleotides such as A3pA and pA3pA are better acceptors than the corresponding [2'-5']-linked compounds, while ImpA2pA is a better donor than ImpA3pA. The reaction between A2pA and ImpA3pA, for example, yields only 4% of product while the reaction of A3pA with ImpA2pA yields 86% of product. The more efficient condensation reactions of dimers are about as efficient as the self-condensation of ImpA. They yield a few percent of material in which five or more substrate molecules are linked together. The percentage of the natural [3'-5']-linkage in the product varies greatly, from as little as 1% to as much as 45%.     

Conformational studies of the smallest structural motifs of DNA detectable via vibrational circular dichroism: cytidylyl-(3''-5'')-guanosine and guanylyl-(3''-5'')-cytidine.

The infrared absorption and vibrational circular dichroism (VCD) spectra of buffered aqueous solutions of cytidylyl-(3'-5')-guanosine (5'(CG)3') and guanylyl-(3'-5')-cytidine (5'(GC)3') are reported. Under low ionic strength conditions, these dinucleotides exhibit VCD features that can be predicted qualitatively from structural data of (CG)2 and (GC)2 sequences of poly(dG-dC).poly(dG-dC), using the exciton model for infrared VCD intensities.     

A gamma-pyronyl-triterpenoid saponin from Pisum sativum.

A new triterpenoid saponin was isolated from Pisum sativum and characterized as 3-O-[alpha-L-rhamnopyranosyl-(1----2)-beta-D-galactopyranosyl(1----2)-be ta- D-glucuronopyranosyl(1----)]-22-O-[3'-hydroxy-2'-methyl-5',6'-dihy dro-4'- pyrone(6'----)]-3 beta, 22 beta, 24-trihydroxyolean-12-ene. The name chromosaponin I is proposed. Chromosaponin I yielded soyasaponin I, known as phytochrome inhibitor, during extraction, but the latter was not found in the free form in this plant.     

Oligonucleotide-europium complex conjugate designed to cleave the 5' cap structure of the ICAM-1 transcript potentiates antisense activity in cells

The 5' cap structure of mRNA is a N7 methylated guanosine residue that is linked by a 5'-5' triphosphate linkage to the 5'-terminus of cellular and viral RNAs synthesized by RNA polymerase II. This unique structure facilitates several processes of mRNA metabolism, including splicing, nucleocytoplasmic transport,initiation of translation, and degradation. Previous research has demonstrated that the lanthanide macrocycle complex, Eu(THED)3+, effectively cleaves the 5' cap structure of mRNA in solution by nucleophilic attack of the triphosphate linkage via the metal-activated hydroxyethyl group of the THED ligand. This report shows that attachment of a Eu(THED)3+analog to the 3'-terminus of an antisense oligonucleotide, which targets the 5'-terminus of the intercellular adhesion molecule 1 mRNA, potentiates the inhibitory activity of the antisense oligonucleotide in cytokine-treatedendothelial cells.     

Growth-Promoting Action of Adenosine-Containing Dinucleotide on Neuroblastoma Cells: Detection of Adenosine-Cytidine Dinucleotide (ApCp) in Neurofibroma (NF1) Extracts

Abstract: Neurofibroma type 1 tissue was investigated for the presence of growth-promoting activity on human neuroblastoma cells. The activity was isolated by gel filtration and reversed-phase column chromatographs from neurofibroma type 1 extracts. An adenosine-containing dinucleotide (adenylyl(3'-5')cytidine-3'-phosphate) was identified as one of the major components of the activities by its enzymatic fragmentation and liquid chromatography/mass spectrometry. Synthetic adenosine-containing dinucleotide derivatives such as cytidyl(3'-5')adenosine, cytidyl(2'-5')adenosine, adenylyl(3'-5')cytidine, and adenylyl(2'-5')cytidine showed a similar action. Cytidyl(3'-5')adenosine, cytidyl(2'-5')adenosine, and adenylyl(2'-5')cytidine, which are able to release a free adenosine through enzymatic hydrolysis, in particular elicited a strong activity corresponding to that of adenosine with the highest action. These results suggest that neuroblastoma cells are able to use adenosine-containing dinucleotides as well as mononucleotides for their survival and proliferation.     

Conformational studies of (2''-5'') polynucleotides: theoretical computations of energy, base morphology, helical structure, and duplex formation.

A detailed theoretical analysis has been carried out to probe the conformational characteristics of (2'-5') polynucleotide chains. Semi-empirical energy calculations are used to estimate the preferred torsional combinations of the monomeric repeating unit. The resulting morphology of adjacent bases and the tendency to form regular single-stranded structures are determined by standard computational procedures. The torsional preferences are in agreement with available nmr measurements on model compounds. The tendencies to adopt base stacked and intercalative geometries are markedly depressed compared to those in (3'-5') chains. Very limited families of regular monomerically repeating single-stranded (2'-5') helices are found. Base stacking, however, can be enhanced (but helix formation is at the same time depressed) in mixed puckered chains. Constrained (2'-5') duplex structures have been constructed from a search of all intervening glycosyl and sugar conformations that form geometrically feasible phosphodiester linkages. Both A- and B-type base stacking are found to generate non-standard backbone torsions and mixed glycosyl/sugar combinations. The 2'- and 5'-residues are locked in totally different arrangements and are thereby prevented from generating long helical structures.     

Diadenosine 5',5"'-P1,P4-tetraphosphate and related adenylylated nucleotides in Salmonella typhimurium

Salmonella typhimurium LT2 rapidly accumulates high levels of a family of five adenylylated nucleotides following exposure to a bacteriostatic quinone, 6-amino-7-chloro-5,8-dioxoquinoline. These compounds have been analyzed using our recently described two-dimensional thin layer chromatographic method. The five dinucleotides, which cannot be detected in exponentially growing cells, have been identified as diadenosine 5',5"'-P1,P4-tetraphosphate (AppppA), ApppGpp (guanosine 3'-diphosphate-5'-adenosine-5'-(P1,P3-triphosphate)), AppppG (adenosine 5'-guanosine-5'-(P1,P4-tetraphosphate)), ApppG (adenosine 5'-guanosine-5'-(P1,P3-triphosphate)), and ApppA (diadenosine 5',5"'-P1,P3-triphosphate). AppppA has been previously detected in vitro as an enzymatic product of aminoacyl-tRNA synthetases and in vivo at submicromolar levels in eucaryotic cells. The induced intracellular concentration of AppppA and the other adenylylated nucleotides in S. typhimurium is approximately 100-fold higher than that found in eucaryotic cells. We propose that these dinucleotides are alarmones, regulatory molecules signaling a particular metabolic stress.     

Heterogeneity of double-stranded DNA local structure and dynamics: ultrasound studies

A method for studying the local inhomogeneities of DNA and its dynamics is proposed. The method is based on the combination of two procedures, splitting the DNA molecules by ultrasound and analysis of DNA fragments obtained by gel electrophoresis. The frequency of cleavage of internucleotide bonds was found to depend on the type of nucleotides forming the bond and on their nearest neighbors. Estimates of cleavage frequencies in each of 16 dinucleotides showed that, in the 5'-d(CpG)-3', 5'-d(CpA)-3', and 5'-d(CpT)-3' dimers, the cleavage occurs considerably more frequently than in the rest, and the frequency of cleavage depends on the nearest neighbors. It was shown that the double-helix cleavage can occur with shifts by several nucleotides. Physical prerequisites were considered that can lead to this pattern of sequence - specific cleavage.     

Identification and structural characterization of O-beta-ribosyl-(1"----2'')-adenosine-5"-phosphate in yeast methionine initiator tRNA.

We report in this paper on the complete structure determination of the modified nucleotide A*, now called Ar(p), that was previously identified in yeast methionine initiator tRNA as an isomeric form of O-ribosyl-adenosine bearing an additional phosphoryl-monoester group on its ribose2 moiety. By using the chemical procedure of periodate oxidation and subsequent beta-elimination with cyclohexylamine on mono- and dinucleotides containing Ar(p), we characterized the location of the phosphate group on the C-5" of the ribose2 moiety, and the linkage between the two riboses as a (1"----2')-glycosidic bond. Since the structural difference between phosphatase treated Ar(p) and authentic O-alpha-ribosyl-(1"----2')-adenosine from poly(ADP-Ribose) was previously assigned to an isomeric difference in the ribose2-ribose1 linkage, the (1"----2')-glycosidic bond of Ar(p) was deduced to have a beta-spatial configuration. Thus, final chemical structure for Ar(p) at the position 64 in yeast initiator tRNA(Met) has been established as O-beta-ribosyl-(1"----2')-adenosine-5"-phosphate. This nucleotide is linked by a 3',5'-phosphodiester bond to G at the position 65.     

Structures of minor ether lipids isolated from the aceticlastic methanogen, Methanothrix concilii GP6

Structures were determined for two phospholipids and three glycolipids purified from chloroform-methanol extracts of Methanothrix concilii GP6. Together they accounted for 14% of the total lipid and were based on a C20,20-diether core structure consisting of either 2,3-di-O-phytanyl-sn-glycerol or its 3'-hydroxy analog, namely, 2-O-[3,7,11,15-tetramethylhexadecyl]-3-O-[3'- hydroxy-3',7',11',15'-tetramethylhexadecyl]-sn-glycerol. These two core lipids formed phosphodiester bonds to ethanolamine and glycosidic bonds to beta-D-galactopyranose. A third glycolipid consisted of the triglycosyl head group beta-D-galactopyranosyl-(1----6)-[beta-D-glucopyranosyl-(1----3)]-beta-D - galactopyranose in glycosidic linkage to the 3'-hydroxydiether core lipid.     

Inhibition of pancreatic ribonuclease by 2''-5'' and 3''-5'' oligonucleotides.

Forty different oligonucleotides were investigated as possible inhibitors of the depolymerizing activity of RNase A. The strongest inhibitors among the diribonucleoside 2'-5' mono- phosphates were: G2'-5'G, C2'-5'G and U2'-5'G, and among the diribonucleoside 3'-5' monophosphates: ApU, ApC and GpU. Of the eight trinucleotides investigated, ApApUp, ApApCp and ApGpUp were the strongest inhibitors. All four dinucleotides studied (ApUp, ApCp, GpUp and GpCp) were very strong inhibitors, ApUp being the strongest one. The results show that the nature of the various bases in the oligonucleotide has an effect on the degree of inhibition, and that the 3' phosphomonoester group increases the binding of the oligonucleotide to RNase A. These inhibitors can be used in physicochemical and biochemical studies of ribonuclease.     

Synthesis of two phosphate-containing "heptasaccharide" fragments of the capsular polysaccharides of Streptococcus pneumoniae types 6A and 6B.

The "heptasaccharides" O-alpha-D-galactopyranosyl-(1----3)- O-alpha-D-glucopyranosyl-(1----3)-alpha, beta-L-rhamnopyranose 2'-[O-alpha-D-galactopyranosyl-(1----3)-O-alpha-D-glucopyranosyl- (1----3)-O-alpha-L-rhamnopyranosyl-(1----3)-D-ribit-5-yl sodium phosphate] (25) and O-alpha-D-galactopyranosyl- (1----3)-O-alpha-D-glucopyranosyl-(1----3)-alpha, beta-L-rhamnopyranose 2'-[O-alpha-D-galactopyranosyl-(1----3)-O-alpha-D-glucopyranosyl- (1----3)-O-alpha-L-rhamnopyranosyl-(1----4)-D-ribit-5-yl sodium phosphate] (27), which are structural elements of the capsular polysaccharides of Streptococcus pneumoniae types 6A and 6B ([----2)- -alpha-D-Galp-(1----3)-alpha-D-Glcp-(1----3)-alpha-L-Rhap- (1----X)-D-RibOH-(5-P----]n; 6A X = 3, 6B X = 4), respectively, have been synthesized. 2,4-Di-O-acetyl- 3-O-[2,4,6-tri-O-acetyl-3-O-(2,3,4,6-tetra-O-acetyl-alpha-D- galactopyranosyl)-alpha-D-glucopyranosyl]-alpha-L-rhamnopyranosyl trichloroacetimidate (13) was coupled with 5-O-allyloxycarbonyl-1,2,4-tri-O- benzyl-D-ribitol (10), using trimethylsilyl triflate as a promotor (----14), and deallyloxycarbonylation (----15) and conversion into the corresponding triethylammonium phosphonate then gave 16. Condensation of 16 with 4-methoxybenzyl 2,4-di-O-benzyl-3-O-[2,4,6-tri-O-benzyl-3-O-(3,4,6-tri-O-benzyl-alpha-D- galactopyranosyl)-alpha-D-glucopyranosyl]- alpha-L-rhamnopyranoside (22) followed by oxidation and deprotection afforded 25. 5-O-Allyl-1-O-allyloxycarbonyl-2,3-di-O-benzyl-D-ribitol (12) was coupled with 13, using trimethylsilyl triflate as a promoter, the resulting tetrasaccharide-alditol derivative 17 was deallyloxycarbonylated (----18), acetylated (----19), and deallylated (----20), and the product was converted into the triethylammonium phosphonate derivative 21. Condensation of 21 with 22 followed by oxidation and deprotection afforded 27.     

Influence of internucleotide phosphate linkage on relative base stacking in 3'-5' and 2'-5' RNA: a circular dichroic spectroscopic study of RNA hexamer AACCUU.

The variations in base stacking interactions of two isomeric RNA hexamers, 3'-5'r (AACCUU) and 2'-5'r' (AACCUU), have been studied using temperature dependent CD spectroscopy. Both RNA hexamers, in single strand form, exhibited a right handed helical sense. Van't Hoff analysis of the CD spectral results, derived from a two state model, gave a higher enthalpy of stacking for 3'-5' RNA than for 2'-5'RNA. The results suggest that 3'-5' linkage in RNA facilitates formation of better helical stacks in relation to an isomeric 2'-5' linkage.