Secondary ion mass spectrometry: the scope and limitations in structural analysis of oligoribonucleotides

Applicability of secondary ion mass spectrometry (SIMS) for structural analysis of oligoribonucleotides was studied. For this purpose a number of oligoribonucleotides having various base sequences, chain lengths, and internucleotide linkages were prepared, and their positive ion SIMS spectra were recorded without derivatization. The soft ionization produced ions possessing relatively low internal energies which underwent little fragmentation, so that both pseudo-molecular and sequence (fragment) ions were observed, the former being more abundant than the latter. Nevertheless, sufficient information can be obtained to elucidate the structure of oligoribonucleotides. Thus SIMS can be used as a useful method for structural analysis of a wide variety of oligoribonucleotides.     

BRIDGED CYCLIC OLIGORIBONUCLEOTIDES—TOWARDS MODELS FOR CODON-ANTICODON PAIRING

Only three base pairs make up for stable double helices of regular A-type if both helix ends are bridged by flexible non-nucleotide linkers. These cyclic oligoribonucleotides are used as model systems for codon-anticodon pairing in order to reveal base stacking effects arising from structurally relevant bases in the direct neighbourhood of the core triplet duplex.     

Bridged cyclic oligoribonucleotides--towards models for codon-anticodon pairing

Only three base pairs make up for stable double helices of regular A-type if both helix ends are bridged by flexible non-nucleotide linkers. These cyclic oligoribonucleotides are used as model systems for codon-anticodon pairing in order to reveal base stacking effects arising from structurally relevant bases in the direct neighbourhood of the core triplet duplex.     

Application of capillary gas chromatography-mass spectrometry to chemical characterization of radiation-induced base damage of DNA: implications for assessing DNA repair processes

The application of capillary gas chromatography-mass spectrometry (GC-MS) to the chemical characterization of radiation-induced base products of calf thymus DNA is presented. Samples of calf thymus DNA irradiated in N2O-saturated aqueous solution were hydrolyzed with HCOOH, trimethylsilylated, and subjected to GC-MS analysis using a fused-silica capillary column. Hydrolysis conditions suitable for the simultaneous analysis of the radiation-induced products of all four DNA bases in a single run were determined. The trimethylsilyl derivatives of these products had excellent GC properties and easily interpretable mass spectra; an intense molecular ion (M+.) and a characteristic (M-CH3)+ ion were observed. The complementary use of t-butyldimethylsilyl derivatives was also demonstrated. These derivatives provided an intense characteristic (M-57)+ ion, which appeared as either the base peak or the second most intense ion in the spectra. All mass spectra obtained are discussed. Because of the excellent resolving power of capillary GC and the accurate high-sensitivity identification by MS, the capillary GC-MS is suggested as a very suitable technique for identification of altered bases removed from DNA by base excision-repair enzymes such as DNA glycosylases and, thus, as very useful for an understanding of the base excision-repair of DNA.     

The RNA moiety of chick embryo 5-methylcytosine- DNA glycosylase targets DNA demethylation.

We have previously shown that DNA demethylation by chick embryo 5-methylcytosine (5-MeC)-DNA glycosylase needs both protein and RNA. RNA from enzyme purified by SDS-PAGE was isolated and cloned. The clones have an insert ranging from 240 to 670 bp and contained on average one CpG per 14 bases. All six clones tested had different sequences and did not have any sequence homology with any other known RNA. RNase-inactivated 5-MeC-DNA glycosylase regained enzyme activity when incubated with recombinant RNA. However, when recombinant RNA was incubated with the DNA substrate alone there was no demethylation activity. Short sequences complementary to the labeled DNA substrate are present in the recombinant RNA. Small synthetic oligoribonucleotides (11 bases long) complementary to the region of methylated CpGs of the hemimethylated double-stranded DNA substrate restore the activity of the RNase-inactivated 5-MeC-DNA glycosylase. The corresponding oligodeoxyribonucleotide or the oligoribonucleotide complementary to the non-methylated strand of the same DNA substrate are inactive when incubated in the complementation test. A minimum of 4 bases complementary to the CpG target sequence are necessary for reactivation of RNase-treated 5-MeC-DNA glycosylase. Complementation with double-stranded oligoribonucleotides does not restore 5-MeC-DNA glycosylase activity. An excess of targeting oligoribonucleotides cannot change the preferential substrate specificity of the enzyme for hemimethylated double-stranded DNA.     

Rapid identification using pyrolysis mass spectrometry and artificial neural networks of Propionibacterium acnes isolated from dogs

Curie-point pyrolysis mass spectra were obtained from reference Propionibacterium strains and canine isolates. Artificial neural networks (ANNs) were trained by supervised learning (with the back-propagation algorithm) to recognize these strains from their pyrolysis mass spectra; all the strains isolated from dogs were identified as human wild type P. acnes. This is an important nosological discovery, and demonstrates that the combination of pyrolysis mass spectrometry and ANNs provides an objective, rapid and accurate identification technique. Bacteria isolated from different biopsy specimens from the same dog were found to be separate strains of P. acnes , demonstrating a within-animal variation in microflora. The classification of the canine isolates by Kohonen artificial neural networks (KANNs) was compared with the classical multivariate techniques of canonical variates analysis and hierarchical cluster analysis, and found to give similar results. This is the first demonstration, within microbiology, of KANNs as an unsupervised clustering technique which has the potential to group pyrolysis mass spectra both automatically and relatively objectively.     

Nearest-neighbor and next-nearest-neighbor effects in the proton NMR spectra of the oligoribonucleotides ApXpG,CpXpG, CpApXpUpG,ApGpXpC, and ApGpXpCpU

The proton nmr spectra of the oligoribonucleotides in the series CpXpG, ApXpG, CpApXpUpG, and ApGpXpC (X = A, G, C, and U), together with the reference compounds CpG, ApG, CpApUpG, and ApGpC, have been measured. A complete analysis of all the nonexchangeable base protons and the ribose H-1′ protons was made. The insertion of a nucleotide X into a oligoribonucleotide led to shift changes at both nearest-neighbor and next-nearest-neighbor positions, which were rationalized in terms of the shielding abilities of the various bases. The derived shielding trends in the ApGpXpC series of compounds were successfully used to predict the chemical shifts of resonances in the related ApGpXpCpU series.     

Associate hydrations and energies of nucleotide bases as revealed by low-temperature field ionization mass-spectrometric data

The formation of water clusters, polyhydrates of nucleotide bases and their associates during simultaneous condensation of water and base molecules in vacuo onto a surface of a needle emitter cooled to 170 K was studied by field ionization mass spectrometry. It was found that different emitter temperatures are characterized by a specific distribution of intensities of cluster currents, depending on the number of water molecules in clusters. These distributions correlate with structural peculiarities and the relative energetics of formation of water clusters, polyhydrates of nucleotide bases and their associates at low temperature. The features observed in mass spectra for clusters m9Ade (H2O)5, m1Ura (H2O)4 and m9Ade m1Ura (H2O)2 are treated as a result of formation of energetically favorable structures stabilized by H-bonded bridges of water molecules. The relative association constants and formation enthalpies of the noncomplementary pairs Ade Cyt, Gua Ura and the associates which model the aminoacid-base complexes m1Ura Gln and m1.3(2)Thy Gln were determined from the temperature dependencies of the intensities of mass spectra peaks in the range 290-320 K.     

Systematic analysis of desorption chemical ionization (DCI) mass spectra of nucleic acids--7

The positive ion and negative ion pyrolysis mass spectra of the herring sperm DNA have been studied using desorption chemical ionization. The positive ion desorption chemical ionization spectra have been produced with CH4, i-C4H10, NH3, HCl and Cl2; the negative ones with N2O/CH4, N2O/i-C4H10, Cl2, CCl4, HCl and via electron capture. These spectra have been compared with the electron impact ionization spectra. We have observed an important increase of sensitivity when negative ionization has replaced the positive ionization mode. The series of diagnostic ions resulting from direct chemical ionization belong to the family of base + reagent ion X [BH + X] and base + X - HX ion [B]. Their abundance has increased considerably compared to the electron impact spectra. The application of these new diagnostic ions in nucleic acid studies is interesting especially for the much higher abundance of the usually weak dG fragment ion obtained in the negative ionization mode. The dG-base segment of the DNA is the most nucleophilic centre of the whole nucleic acid and is implicated in numerous important biochemical reactions involving, for example, proteins.     

Nearest-neighbor and next-nearest-neighbor effects in the proton NMR spectra of the oligoribonucleotides ApGpX and CpApX

The variable-temperature proton nmr spectra of the oligoribonucleotides in the series CpApX and the series ApGpX, X = A, G, C, U, together with the parent dimers CpA and ApG have been measured. A complete analysis of all the nonexchangeable base proton resonances and ribose H-1′ proton resonances was made. The presence of trends in the shielding abilities of the various bases at both the nearest-neighbor and next-nearest-neighbor positions were identified. The observed shieldings could be used to predict the chemical shifts of protons in related systems. Based on the empirical results from ribodinucleoside monophosphates, the temperature-dependent behavior of the J_1′2′ coupling constants of the triribonucleotides suggested that the compounds in the CpApX series stacked from the 5′-end to the 3′-end, while those in the ApGpX series stacked from the 3′-end to the 5′-end.     

Isolation of sphingoid bases of sea cucumber cerebrosides and their cytotoxicity against human colon cancer cells

Sea cucumber is a health-beneficial food, and contains a variety of physiologically active substances including glycosphingolipids. We show here the sphingoid base composition of cerebrosides prepared from sea cucumber and the cytotoxicity against human colon cancer cell lines. The composition of sphingoid bases prepared from sea cucumber was different from that of mammals, and the major constituents estimated from mass spectra had a branched C17-19 alkyl chain with 1-3 double bonds. The viability of DLD-1, WiDr and Caco-2 cells treated with sea cucumber sphingoid bases was reduced in a dose-dependent manner and was similar to that of cells treated with sphingosine. The sphingoid bases induced such a morphological change as condensed chromatin fragments and increased the caspase-3 activity, indicating that the sphingoid bases reduced the cell viability by causing apoptosis in these cells. Sphingolipids of sea cucumber might therefore serve as bioactive dietary components to suppress colon cancer.     

Isolation of Sphingoid Bases of Sea Cucumber Cerebrosides and Their Cytotoxicity against Human Colon Cancer Cells

Sea cucumber is a health-beneficial food, and contains a variety of physiologically active substances including glycosphingolipids. We show here the sphingoid base composition of cerebrosides prepared from sea cucumber and the cytotoxicity against human colon cancer cell lines. The composition of sphingoid bases prepared from sea cucumber was different from that of mammals, and the major constituents estimated from mass spectra had a branched C17–19 alkyl chain with 1–3 double bonds. The viability of DLD-1, WiDr and Caco-2 cells treated with sea cucumber sphingoid bases was reduced in a dose-dependent manner and was similar to that of cells treated with sphingosine. The sphingoid bases induced such a morphological change as condensed chromatin fragments and increased the caspase-3 activity, indicating that the sphingoid bases reduced the cell viability by causing apoptosis in these cells. Sphingolipids of sea cucumber might therefore serve as bioactive dietary components to suppress colon cancer.     

Advantages of 2'-O-methyl oligoribonucleotide probes for detecting RNA targets.

We have compared various kinetic and melting properties of oligoribonucleotide probes containing 2'-O-methylnucleotides or 2'-deoxynucleotides with regard to their use in assays for the detection of nucleic acid targets. 2'-O-Methyl oligoribonucleotide probes bound to RNA targets faster and with much higher melting temperatures (Tm values) than corresponding 2'-deoxy oligoribonucleotide probes at all lengths tested (8-26 bases). Tm values of both probes increased with length up to approximately 19 bases, with maximal differences in Tm between 2'-O-methyl and 2'-deoxy oligoribonucleotide probes observed at lengths of 16 bases or less. In contrast to RNA targets, 2'-O-methyl oligoribonucleotide probes bound more slowly and with the same Tm to DNA targets as corresponding 2'-deoxy oligoribonucleotide probes. Because of their greatly enhanced Tm when bound to RNA, 2'-O-methyl oligoribonucleotide probes can efficiently bind to double-stranded regions of structured RNA molecules. A 17 base 2'-O-methyl oligoribonucleotide probe was able to bind a double-stranded region of rRNA whereas the same 17 base 2'- deoxy oligoribonucleotide probe did not. Due to their enhanced Tm when bound to RNA targets, shorter 2'-O-methyl oligoribonucleotide probes can be used in assays in place of longer 2'-deoxy oligoribonucleotide probes, resulting in enhanced discrimination between matched and mismatched RNA targets. A 12 base 2'-O-methyl oligoribonucleotide probe had the same Tm as a 19 base 2'-deoxy oligoribonucleotide probe when bound to a matched RNA target but exhibited a much larger decrease in Tm than the 2'-deoxy oligoribonucleotide probe when bound to an RNA target containing either 1 or 2 mismatched bases. The increased Tm, faster kinetics of hybridization, ability to bind to structured targets and increased specificity of 2'-O-methyl oligoribonucleotide probes render them superior to corresponding 2'-deoxy oligoribonucleotides for use in assays that detect RNA targets.     

Mass spectrometric monitoring of solid phase phosphate triester synthesis of DNA fragments

A new and convenient pyrolysis mass spectrometric approach for monitoring solid phase phosphate triester synthesis of oligodeoxyribonucleotides in 5'-3' direction has been developed. The base-typical fragment ions produced by pyrolysis at 280 degrees C of the polymer-bound oligonucleotide triesters in the mass spectrometer permit the analytical monitoring of DNA chain growth, using simple mathematical operations. The base composition can be determined directly from the polymer. In addition, minor nucleosides can easily be detected.     

Labile protecting groups in tRNA synthesis

The use of labile protecting groups for the protection of the exocyclic amino function of adenine, guanine and cytosine has two main advantages in RNA synthesis. Final deprotection in concentrated aqueous ammonia takes place in milder conditions which are more compatible with the sensitivity of oligoribonucleotides towards alkali-conditions. The introduction of fragile bases such as certain modified bases encountered in the primary structure of t-RNA is feasible. The chemical synthesis of RNA fragments constituting the primary structure of B. subtilis f-methionine t-RNA is described.     

Physical consequences of chemical modification: circular dichroism of the kethoxalated oligoribonucleotides

Alterations in CD spectra are found in G-containing oligoribonucleotides after modification with kethoxal (beta-ethoxy--alpha-ketobutyraldehyde). Stacking interactions in kethoxalated oligomers are followed by temperature dependence of their CD amplitudes. It is shown that for oligomers with nucleosides in anti-conformation adduct formation destroys the stacking interaction with 3'-neighbour but not with a 5'-neighbour. For nucleosides in non-standard conformation (i.e. syn-conformation of guanine in GpGpCp) the physical alteractions may be seen in those cases, when the substituting group affects the initial conformation or the interplane base contacts via, for instance, blocking NH(2)-group of guanine in GpUp.The results demonstrated that even a single monomer modification in a polymer chain could not be considered as a local event having no influence on the three-dimensional structure. The degree of conformational disorders depends both on the conformation of single nucleotides in the stack and on the nature of the nearest neighbours of the modified base.     

Raman spectroscopic measurement of base stacking in solutions of adenosine, AMP, ATP, and oligoadenylates

Measurements of the colligative properties of nucleosides and their derivatives have shown that bases form transient aggregates in solution [Ts'o (1967) J. Am. Chem. Soc. 89, 3612-3622]. Aggregation of nucleotides cannot be measured by osmometry due to the presence of counterions. Sedimentation measurements are difficult to obtain and have been complicated by differences in pH [Ferguson et al. (1974) Biophys. Chem. 1, 325-337]. Raman studies of oligonucleotides have shown that the intensities due to base vibrational modes depend on the extent of base stacking, but this dependence has not been quantitated. We have measured this dependence by relating changes in the Raman spectra of nucleotides and nucleosides with previous measurements of colligative properties. Visible Raman spectra of ATP, AMP, and adenosine, taken over a range of concentrations from 1 to 1000 mM, show that the peak intensity ratio (I1305 + I1380)/I1340 varies linearly with the log of the concentration for all three bases. This concentration-dependent change correlates with published molal osmotic coefficient data for functionally similar bases with a correlation coefficient of 0.99. In contrast, UV resonance Raman spectra of the same bases show changes that vary linearly with concentration.     

Thermodynamics of double- and triple-helical aggregates formed by self-complementary oligoribonucleotides of the type rAxUy

The thermal denaturation of a series of oligoribonucleotides of the form rAxUy (x = 5 or 7 and y = 3-11) has been characterized by means of IR spectroscopy, UV spectroscopy, and DSC. IR spectra proved the occurrence of double- and triple-helical regions at various contents of uracil residues in the nucleotide. From DSC measurements transition enthalpies, entropies, and free enthalpies were derived. The effect of fraying in terminal base pairs of symmetrical nucleotides (x = y) was quantified. Thermodynamic excess parameters due to dangling ends (5'A and 3'U), terminal AU base pairs, and UAU base triplets were obtained by comparing DSC results from different nucleotides. Empirical values for contributions of base stacking and pairing to the stability of terminal AU base pairs have been estimated: for nucleotides under study with a high degree of fraying at the ends of the helix the major stabilization effect comes from base stacking. The size of the cooperative unit lambda in most nucleotides under study is larger than 1; i.e., in these cases intermolecular cooperation takes place. Through deconvolution of DSC data maximum populations of intermediate states FI,max were obtained. On the basis of these results all nucleotides under study were proved to melt in multistate manner. FI,max increases with the number of base pairs, decreases through dangling ends, and shows approximately constant values for triple-helical aggregates of the series rA5Uy as well as rA7Uy.