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PubMed1984

[Primary structure of tRNA2Leu of bovine mammary gland. Oligonucleotides of T1-RNAse hydrolysate. Reconstruction of a total nucleotide sequence].

The oligonucleotides obtained by digestion of tRNA2Leu from cow mammary gland with T1 RNase were separated by micro-column chromatography on DEAE-cellulose in 7 M urea at pH 7,5 and 3,7, and in addition on Dowex 1 x 2. The digest consisted of 18 individual components, the larger being a tridecanucleotide. Micro-column chromatography of nucleotides on anion-exchanger AG 1 x 8 and nucleosides on Aminex A-6 was used to determine the base composition of the oligonucleotides. The oligonucleotide structure was established using terminal analysis, hydrolysis by pancreatic and U2-RNases and incomplete hydrolysis by snake venom phosphodiesterases. The total primary structure of tRNA2Leu was derived from overlapping fragments isolated after its complete hydrolysis with pancreatic and T1 RNase and using data obtained on S1-nuclease digestion of tRNA. The methods of rapid gel-sequencing were also employed for checking the nucleotide sequence of tRNA2Leu from cow mammary gland.

Animals↗
PubMed1996

Investigation of the intracellular stability and formation of a triple helix formed with a short purine oligonucleotide targeted to the murine c-pim-1 proto-oncogene promotor.

In our previous work we have shown that the oligonucleotide 5'-GGGGAGGGGGAGG-3' gives a very stable and specific triplex with the promoter of the murine c-pim-1 proto-oncogene in vitro[Svinarchuk, F., Bertrand, J.-R. and Malvy, C.(1994)Nucleic Acids Res., 22, 3742-3747]. In the present work, we have tested triplex formation with some derivatives of this oligonucleotide which are designed to be degradation-resistant inside the cells, and we show that phosphorothioate and the oligonucleotide with a 3' terminal amino group are still able to form triplexes. Moreover these oligonucleotides, like the 13mer oligonucleotide of similar composition [Svinarchuk, F., Paoletti, J., and Malvy, C. (1995) J. Biol. Chem., 270, 14068-14071], are able to stabilize the targeted duplex. In vivo DMS footprint analysis after electroporation of the pre-formed triplex into the cell have shown the presence of the triple helix inside the cells. This triplex structure partially blocks c-pim-1 promotor activity as shown by transient assay with a c-pim-1 promoter-luciferase gene construct. To our knowledge these data are the first direct evidence that conditions inside cells are favorable for triplex stability with non-modified oligonucleotides. However we were unable to show triplex formation inside living cells using various methods of oligonucleotide delivery. We suppose that this may be due to the oligonucleotide being sequestered by cellular processes or proteins. Further work is needed to find oligonucleotide derivatives and ways of their delivery to overcome the problem of triplex formation inside the cells.

Animals↗
PubMed2005

Unusual chromatographic behavior of oligonucleotide sequence isomers on two different anion exchange HPLC columns.

The retention behavior of the unmodified phosphodiester oligonucleotide sequence isomers was investigated on two different anion exchange columns: Biospher GMB 1000Q (based on DEAE-modified glycidyl methacrylate) and PolyWAX LP (based on silica with a crosslinked coating of linear polyethyleneimine). There was a notable difference in retention of oligonucleotides of the same composition but differing in the position of a single base. The most pronounced difference was observed between the oligonucleotides with the variable base in the end and in the center of the sequence. The use of either acetonitrile or 2-propanol as a mobile phase organic modifier did not markedly affect the retention time patterns. Prediction of the retention times of oligonucleotides must take into account the base position as well as identity. This is the first report of such a "same composition different sequence" effect, described for the short peptides, for synthetic oligonucleotides.

2-Propanol↗
PubMed1986

Specific-primer-directed DNA sequencing.

A simple and rapid strategy for DNA sequence analysis based on the Sanger chain-termination method is described. This procedure utilizes full-sized inserts of 1 to 4 kb of DNA cloned into M13 bacteriophage vectors. After the sequence of the first 600-650 bp of the insert DNA has been determined with the commercially available universal vector primer, a specific oligonucleotide is synthesized utilizing the sequence data obtained from the 3' end of the sequence and used as a primer to extend the sequence analysis for another 600-650 nucleotides. Additional primers are synthesized in a similar manner until the nucleotide sequence of the entire insert DNA has been determined. General guidelines for the selection of oligonucleotide length and composition and the use of unpurified primers are discussed. The use of the specific-primer-directed approach to dideoxynucleotide sequence analysis, in association with highly purified single-stranded template DNA, reduces considerably the time required for the analysis of large segments of DNA.

Bacteriophages↗
PubMed2004

Automated de novo sequencing of nucleic acids by liquid chromatography-tandem mass spectrometry.

We present the first global computer-aided sequencing algorithm for the de novo determination of short nucleic acid sequences. The method compares the fragment ion spectra generated by collision-induced dissociation of multiply charged oligodeoxynucleotide-ions to the m/z values predicted employing established fragmentation pathways from a known reference sequence. The closeness of matching between the measured spectrum and the predicted set of fragment ions is characterized by the fitness, which takes into account the difference between measured and predicted m/z values, the intensity of the fragment ions, the number of fragments assigned, and the number of nucleotide positions not covered by fragment ions in the experimental spectrum. Smaller values for the fitness indicate a closer match between the measured spectrum and predicted m/z values. In order to find the sequence most closely matching the experimental spectrum, starting from a given nucleotide composition all possible oligonucleotide sequences are assembled followed by identification of the correct sequence by the lowest fitness value. Using this concept, sequences of 5- to 12-mer oligodeoxynucleotides were successfully de novo determined. High sequence coverage with fragment ions was essential for obtaining unequivocal sequencing results. Moreover, the collision energy was shown to have an impact on the interpretability of tandem mass spectra by the de novo sequencing algorithm. Experiments revealed that the optimal collision energy should be set to a value just sufficient for complete fragmentation of the precursor ion.

Algorithms↗
PubMed2006

Electrochemistry using self-assembled DNA monolayers on highly oriented pyrolytic graphite.

Duplex DNA functionalized with pyrene has been utilized to fabricate DNA-modified electrodes on highly oriented pyrolytic graphite (HOPG). Films have been characterized using AFM and radioactive labeling as well as electrochemically. The data obtained are consistent with a close-packed structure in the film with helices oriented in a nearly upright orientation, as seen earlier with the fabrication of thiol-tethered duplexes on gold. Also as on gold, we observe the reduction of DNA-bound intercalators in a DNA-mediated reaction. The reduction of the intercalator is attenuated in the presence of the single-base mismatches, CA and GT, independent of the sequence composition of the oligonucleotide. This sensitivity to single-base mismatches is enhanced when methylene blue reduction is coupled in an electrocatalytic cycle with ferricyanide. The extended potential range afforded by the HOPG surface has allowed us also to investigate the electrochemistry of previously inaccessible metallointercalators, Ru(bpy)2dppz2+ and Os(phen)2dppz2+, at the DNA-modified HOPG surface. These results support the application of DNA-modified HOPG as a convenient and reproducible surface for electrochemical DNA sensors using DNA-mediated charge transport.

Cross-Linking Reagents↗
PubMed1965

METHODS FOR AUTOMATIC NUCLEOTIDE-SEQUENCE ANALYSIS. MULTICOMPONENT SPECTROPHOTOMETRIC ANALYSIS OF MIXTURES OF NUCLEIC ACID COMPONENTS BY A LEAST-SQUARES PROCEDURE.

1. A theoretical analysis of the errors in multicomponent spectrophotometric analysis of nucleoside mixtures, by a least-squares procedure, has been made to obtain an expression for the error coefficient, relating the error in calculated concentration to the error in extinction measurements. 2. The error coefficients, which depend only on the ;library' of spectra used to fit the experimental curves, have been computed for a number of ;libraries' containing the following nucleosides found in s-RNA: adenosine, guanosine, cytidine, uridine, 5-ribosyluracil, 7-methylguanosine, 6-dimethylaminopurine riboside, 6-methylaminopurine riboside and thymine riboside. 3. The error coefficients have been used to determine the best conditions for maximum accuracy in the determination of the compositions of nucleoside mixtures. 4. Experimental determinations of the compositions of nucleoside mixtures have been made and the errors found to be consistent with those predicted by the theoretical analysis. 5. It has been demonstrated that, with certain precautions, the multicomponent spectrophotometric method described is suitable as a basis for automatic nucleotide-composition analysis of oligonucleotides containing nine nucleotides. Used in conjunction with continuous chromatography and flow chemical techniques, this method can be applied to the study of the sequence of s-RNA.

Adenine↗
PubMed1998

Identification of the reaction products of (2'-5')oligoadenylate synthetase in the marine sponge.

Previously we reported on the presence of a high (2'-5')oligoadenylate synthetase activity in the marine sponge Geodia cydonium [Kuusksalu, A., Pihlak, A., Müller, W. E. G. & Kelve, M. (1995) Eur. J. Biochem. 232, 351-357]. The presence of (2'-5')oligoadenylates [(2'-5')A] in crude sponge extract was shown by radioimmunoassay and by their HPLC comigration with authentic (2'-5')A oligomers. In addition, the sponge (2'-5')oligoadenylates displayed biological activity, as determined by inhibition studies of protein biosynthesis in rabbit reticulocyte lysate. In the present study individual (2'-5')oligoadenylates synthesized by sponge enzyme were separated by HPLC. The exact composition of every oligonucleotide peak eluted was determined by matrix-assisted laser-desorption-ionization mass spectrometry (MALDI-MS) analysis. The 2'-5' phosphodiester bond in oligoadenylates was verified by NMR analysis. Based on the high concentration of (2'-5')A oligomers in G. cydonium and their similarity with those found in mammals we propose that the (2'-5')A system is involved in a cytokine-mediated pathway and/or in a protection system against viruses, present in the marine environment.

2',5'-Oligoadenylate Synthetase↗
PubMed1995

Purification of the spliceosome A-complex and its visualization by electron microscopy.

Pre-mRNA splicing occurs on spliceosomes, a family of ribonucleoprotein particles. Spliceosome assembly on exogenous adenovirus pre-mRNA was blocked at the A-complex (or pre-spliceosome) stage, either by destruction of the small nuclear ribonucleoproteins (snRNPs) that comprise the U4/U5/U6 tri-snRNP complex, or by interference in tri-snRNP assembly and interactions. The A-complex was isolated by size exclusion chromatography; homogeneity was shown by electrophoresis in nondenaturing polyacrylamide gels, gradient sedimentation, and electron microscopy. Northern hybridization showed U1 and U2 snRNAs to be present in the preparation, but not U4, U5, or U6. Antibodies specific for a component of the U1 snRNP or for a component that is common to all snRNPs (except U6) each precipitated an A-complex containing pre-mRNA, U1 and U2 snRNPs. Electron micrographs showed 230 x 270-A particles whose two components appear similar to individual U1 and U2 snRNPs. Electron micrographs of an A-complex-5'-biotinyl oligonucleotide-streptavidin-gold composite allowed identification of the U2 snRNP within the structure and the localization of the 5'-segment of U2 snRNA at a unique site in the A-complex. This region of U2 RNA is adjacent to the developing catalytic center of the spliceosome.

Base Sequence↗
PubMed2004

Ribonucleic acids of human milk.

The milk feeding is the most essential process laying the foundation of human health at the postnatal development. However little is known about nucleic acids secreted into mother's milk during lactation. In order to investigate the composition and abundance of human milk NA we adapted the conventional isolation method to achieve high yield of total nucleic acids from milk samples. Concentration of total NA in milk samples of different donors varies from 20 to 68 mkg/ml at early stages of lactation. The average concentration tends to fall down to the end of lactation. The chain length of the major forms of NA varies from mononucleotides up to approximately 100 bases. Compositions of milk oligonucleotides are similar in samples of different donors. Major milk oligonucleotides are formed of RNA. Human milk contains the set of long-chain oligonucleotides with a developed secondary structure. Sequences of some oligo-RNAs correspond to the 3'-part of 5.8 S human ribosomal RNA and to the 3'-parts of tRNAVal and tRNATyr Primary structures of some others oligo-RNAs were related to fragments of human 18S and 28S rRNAs.

Base Sequence↗
PubMed2002

Oligonucleotide fingerprinting of rRNA genes for analysis of fungal community composition.

Thorough assessments of fungal diversity are currently hindered by technological limitations. Here we describe a new method for identifying fungi, oligonucleotide fingerprinting of rRNA genes (OFRG). ORFG sorts arrayed rRNA gene (ribosomal DNA [rDNA]) clones into taxonomic clusters through a series of hybridization experiments, each using a single oligonucleotide probe. A simulated annealing algorithm was used to design an OFRG probe set for fungal rDNA. Analysis of 1,536 fungal rDNA clones derived from soil generated 455 clusters. A pairwise sequence analysis showed that clones with average sequence identities of 99.2% were grouped into the same cluster. To examine the accuracy of the taxonomic identities produced by this OFRG experiment, we determined the nucleotide sequences for 117 clones distributed throughout the tree. For all but two of these clones, the taxonomic identities generated by this OFRG experiment were consistent with those generated by a nucleotide sequence analysis. Eighty-eight percent of the clones were affiliated with Ascomycota, while 12% belonged to BASIDIOMYCOTA: A large fraction of the clones were affiliated with the genera Fusarium (404 clones) and Raciborskiomyces (176 clones). Smaller assemblages of clones had high sequence identities to the Alternaria, Ascobolus, Chaetomium, Cryptococcus, and Rhizoctonia clades.

DNA Fingerprinting↗
PubMed1997

Interaction of oligonucleotides with barrier fluid proteins.

Affinity modification of proteins was used to study their interaction with oligonucleotides barrier fluids. Several proteins of saliva and tears were shown to undergo affinity modification during incubation with an alkylating derivative of deoxyribooligonucleotides. In tears, such proteins were lactoferrin, immunoglobin G and lysozyme; in saliva, immunoglobulin A and lysozyme. The data showed that the affinity for oligonucleotides decrease in the order lactoferrin > lysozyme > immunoglobulin A > immunoglobulin G. The binding of reactive oligonucleotide derivatives with the proteins was competitively inhibited by polyanions, such as oligonucleotides of various nucleotide compositions, single-stranded and double-stranded DNA, heparin, and dextran sulfate. Interactions between oligonucleotides and proteins can strongly affect the metabolism of oligonucleotides and their ability to permeate biological barriers.

Adenosine Triphosphate↗
PubMed1997

Selectivity of anion exchange chromatography and capillary gel electrophoresis for the analysis of phosphorothioate oligonucleotides.

The complementary nature of anion exchange chromatography and capillary gel electrophoresis for oligonucleotide analysis is demonstrated by evaluating a comprehensive series of authentic deletion sequences and partial phosphodiester analogs of five phosphorothioate oligonucleotides of different base composition and sequence. While anion exchange HPLC is sensitive to differences in backbone length of phosphorothioate oligonucleotides, oligomers with length difference of one base unit are not resolved. Capillary gel electrophoresis, on the other hand, has excellent single-base resolution while being relatively insensitive to phosphate in the phosphorothioate backbone. The data definitively establish the necessity of employing both separation techniques for adequate characterization of lower order process-related impurities potentially found in synthetic phosphorothioate oligonucleotides.

Chromatography, Ion Exchange↗
PubMed1991

Chemical synthesis of oligonucleotides containing a naphthalene diolepoxide deoxycytidine adduct in solution and using a mixed chemistry semi-automated solid phase approach.

I have recently reported the first total synthesis of a naphthalene diolepoxide--deoxycytidine nucleoside adduct suitable for incorporation into synthetic oligonucleotides. The racemic synthesis yielded the modified deoxycytidine nucleoside as a mixture of diastereomers (1a,b). I now report the conversion of the modified nucleosides (1a,b) into their phosphotriester salts (2a,b) and subsequent incorporation at a specific site into synthetic oligodeoxyribonucleotides. These syntheses yielded oligonucleotides possessing the diastereomerically modified nucleosides, N4-[(+/-)-1(b),2(a),3(a)-trihydroxy-4(b)-1,2,3,4-tetrahydronaphthyl]-2'- deoxycytidine (3a,b = C*), at a specific position. A sample of the modified nucleosides (3a,b) was obtained by chemically removing the protecting groups from (1a,b) and purification on C-18 reverse-phase chromatography. The structure of the modified nucleosides (3a,b) was supported by 400 MHz 1H-NMR and fast atom bombardment MS. The modified tetramers C*pGpApT (9a,b) along with the parent sequence CpGpApT were synthesized using the phosphotriester chemistry in solution. Interestingly, the diastereomerically modified tetramers (9a) and (9b) could be resolved using C-18 reverse-phase chromatography. Using a mixed chemistry semi-automated approach on a solid support the modified oligonucleotide 24mers, ApApTpTpGpCpApApGpTpC*pCpApTpApTpGpGpApCpTpTpGpC (10a,b), were synthesized and purified by anion exchange chromatography. The nucleoside composition of all synthetic oligonucleotides was quantitatively examined by reverse-phase chromatography following enzymic digestion.

Base Sequence↗
PubMed2002

Synthesis and evaluation of phosphorescent oligonucleotide probes for hybridisation assays.

Monofunctional, p-isothiocyanatophenyl-derivatives of platinum (II)-coproporphyrin-I (PtCP-NCS) were evaluated as phosphorescent labelling reagents for synthetic oligonucleotides containing a 3'- or 5'-amino modification. Synthesis and purification conditions were optimised to generate high yields and purity of PtCP-labelled oligonucleotide probes. Phosphorescent properties of the PtCP label have been shown to be largely unaffected by conjugation to oligonucleotides of various length, GC composition and label attachment site. 5'-PtCP-labelled oligonucleotides were shown to work efficiently as primers in a standard PCR. A dedicated 532 nm laser-based time-resolved fluorescence plate reader enabled highly sensitive detection of PtCP-labelled oligonucleotides and PCR products, both in solution and in agarose gels, with limits of detection in the order of 0.3 pM. A model system employing two complementary oligonucleotides labelled with PtCP and QSY 7 dye (dark quencher) showed strong (approximately 20-fold) and specific proximity quenching of PtCP label upon hybridisation in solution. The potential applications of PtCP-labelled probes in hybridisation assays were discussed.

Luminescent Measurements↗
PubMed1994

Electron migration in oligonucleotides upon gamma-irradiation in solution.

Electron migration in irradiated solutions of DNA was investigated using 5-bromouracil synthetically incorporated into oligonucleotides of defined base composition as a molecular indicator of electron interactions. Solvated electrons interact quantitatively with 5-bromouracil, leading to a highly reactive 5-yl radical which can abstract an adjacent hydrogen atom to yield uracil. Yields of uracil, or loss of 5-bromouracil, from irradiated oligonucleotide samples were measured using gas chromatography-mass spectrometric analysis of their trimethylsilylated acid hydrolysates. To examine the effects of base composition and DNA conformation on electron migration, a set of oligonucleotides containing 5-bromouracil at selected positions with three base (guanine, cytosine, thymine or adenine) spacers (e.g. [BrU(GGG)3]3) were irradiated in their single- or double-stranded form following annealing with appropriate complementary sequences. Differences in uracil yields suggested that electron migration occurred to different extents in oligonucleotides containing different base sequences. In irradiated single-stranded oligonucleotides, the yield of uracil decreased in the order A > T > > C approximately G. However, in irradiated double-stranded oligonucleotides, the yield of uracil decreased in the order G > C approximately T > A. These differences were attributed to proton-transfer reactions facilitated by base pairing in double-stranded oligonucleotides. The distance over which the electron would migrate was then determined using a series of oligonucleotides containing 5-bromouracil at selected positions with guanine spacers (i.e. [BrU(G)n]3 (n = 3, 5, 7, 9). Oligonucleotides were irradiated in their double-stranded form following annealing with the appropriate complementary sequences. Analysis of the loss of 5-bromouracil revealed that electron migration occurred efficiently over c. 3-4 guanine bases assuming that migration could occur as efficiently in either direction along the DNA molecule. These data can be compared with studies reporting more extensive migration for electrons generated by direct ionization of DNA.

Base Sequence↗
PubMed1996

Synthesis and thermodynamics of oligonucleotides containing chirally pure R(P) methylphosphonate linkages.

Methylphosphonate (MP) oligodeoxynucleotides (MPOs) are metabolically stable analogs of conventional DNA containing a methyl group in place of one of the non-bonding phosphoryl oxygens. All 16 possible chiral R(P) MP dinucleotides were synthesized and derivatized for automated oligonucleotide synthesis. These dimer synthons can be used to prepare (i) all-MP linked oligonucleotides having defined R(P) chirality at every other position (R(P) chirally enriched MPOs) or (ii) alternating R(P) MP/phosphodiester backbone oligonucleotides, depending on the composition of the 3'-coupling group. Chirally pure dimer synthons were also prepared with 2'-O-methyl sugar modifications. Oligonucleotides prepared with these R(P) chiral methylphosphonate linkage synthons bind RNA with significantly higher affinity than racemic MPOs.

Chromatography, High Pressure Liquid↗
PubMed1998

Induced circular dichroism of benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide stereoisomers covalently bound to deoxyribooligonucleotides used to probe equilibrium distribution between groove binding and intercalative adduct conformations.

Binding conformations of single anti-BPDE-N2-dG adducts in oligonucleotides of varying base composition have been studied by induced circular dichroism (ICD). The sign of the ICD around 350 nm of single-stranded oligonucleotide adducts and the sign of an exciton type of CD component at 260 nm in both single strand and duplex forms of adducts correlate with the absolute configuration of the cyclohexyl moiety of the adduct. Changes in magnitude and sign of the ICD around 350 nm were observed upon duplex formation. The results show that adducts displaying external (minor groove) binding characteristics are associated with a significant positive ICD. Conversely, adducts displaying intercalation binding characteristics were found to have a positive or negative ICD. The magnitude of the ICD is dependent on the sequence context and the particular adduct isomer studied. Duplexes with (+)-trans-anti-BPDE-N2-dG in 5'-d(CCTATCGCTATCC) or 5'-d(CCTATAGATATCC) exhibit a relatively strong positive ICD. In contrast, the duplexes with (+)-trans-anti-BPDE-N2-dG in 5'-d(CCTATTGCTATCC) and 5'-d(CCTATTGTTATCC) display a small positive and negative ICD, respectively, in both cases suggesting conformational heterogeneity. Partially complementary duplexes (dA, dT, or dG) localized opposite the (+)-trans-anti-BPDE-N2-dG adduct in 5'-d(CCTATCGCTATCC) or 5'-d(CCTATAGATATCC) also demonstrated negative ICD. These results together with light absorption characteristics suggest a preferred conformation of intercalation for the mismatched duplexes. Evidence of an equilibrium between the external and intercalative adduct conformation is provided by the results from the temperature dependence of the near-UV absorption and ICD characteristics of (+)-trans-anti-BPDE-N2-dG complex in a 5'-d(CCTATAGATATCC) duplex.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗