共查询到20条相似文献,搜索用时 31 毫秒
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One major difficulty in the analysis of nucleic acids by electrospray mass spectrometry is represented by the affinity of the polyanionic sugar-phosphate backbone for nonvolatile cations, especially ubiquitous sodium and potassium ions. A simple on-line sample preparation system comprising a microflow pumping system and 45 x 0.8-mm-i.d. microcolumns packed with weak or strong cation-exchange resins is described for the efficient removal of cations from nucleic acid samples. Samples were analyzed by flow injection analysis at a 3-5 microL/min flow of 10 mM triethylamine in 50% water-50% acetonitrile. After on-line desalting, mass spectra of oligonucleotides revealed no significant sodium adduct peaks. Moreover, signal-to-noise ratios were greatly enhanced compared to direct injection of the samples. Electrospray mass spectrometry with on-line sample preparation allowed accurate molecular mass determinations of picomole amounts of crude oligonucleotide preparations ranging in size from 8 to 80 nucleotides within a few minutes. The good linearity of the calibration plot (R2 = 0.9988) over at least 2 orders of magnitude and a relative standard deviation in peak areas of less than 9% permitted the sensitive quantitative measurement of oligonucleotides in a concentration range of 0.2-20 microM with selected-ion monitoring. Finally, the on-line sample preparation system was evaluated for the mass spectrometric analysis of complex oligonucleotide mixtures. 相似文献
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V Zecchini D Caucheteux J Grétry M Lema?tre J Dommès 《Canadian Metallurgical Quarterly》1995,19(2):286-290
This article describes the optimization of the hybridization signal obtained with biotinylated oligonucleotides. Optimal number and positions of biotin moieties on a 33-base oligonucleotide probe were determined. The quality of avidin-peroxidase conjugate and the choice of chromogenic substrate influenced detection sensitivity. A signal amplification method was also developed for avidin enzymatic conjugates. These improvements allowed the detection of less than 0.02 fmol of target DNA. 相似文献
4.
The use of uniformly 13C,15N-labeled RNA has greatly facilitated structural studies of RNA oligonucleotides by NMR. Application of similar methodologies for the study of DNA has been limited, primarily due to the lack of adequate methods for sample preparation. Methods for both chemical and enzymatic synthesis of DNA oligonucleotides uniformly labeled with 13C and/or 15N have been published, but have not yet been widely used. We have developed a modified procedure for preparing uniformly 13C,15N-labeled DNA based on enzymatic synthesis using Taq DNA polymerase. The highly efficient protocol results in quantitative polymerization of the template and approximately 80% incorporation of the labeled dNTPs. Procedures for avoiding non-templated addition of nucleotides or for their removal are given. The method has been used to synthesize several DNA oligonucleotides, including two complementary 15 base strands, a 32 base DNA oligonucleotide that folds to form an intramolecular triplex and a 12 base oligonucleotide that dimerizes and folds to form a quadruplex. Heteronuclear NMR spectra of the samples illustrate the quality of the labeled DNA obtained by these procedures. 相似文献
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Reported here is how the hybridization of individual components of oligonucleotide mixtures to solid-phase bound complementary strands can be monitored simultaneously by quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Three oligonucleotides, a DNA heptamer, a DNA octamer and a DNA octamer with a terminal cholic acid appendage were used as the test mixture. Upon cooling in the presence of a complementary undecamer on controlled pore glass, depletion of the components from the solution was observed. The resulting hybridization curves show the same relative affinities as traditional UV melting curves with single components and their complement. Assays of the kind described here may be used to select high affinity binders from combinatorial libraries of modified antisense oligonucleotides. 相似文献
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J Frank DK Kelleher A Pompella O Thews HK Biesalski P Vaupel 《Canadian Metallurgical Quarterly》1998,58(13):2693-2698
Polymerase chain reaction (PCR) assays were designed to amplify 56- and 99-base regions of the pmoA gene from Methylosinus trichosporium OB3b and Methylomicrobium albus BG8, two species of methanotrophic bacteria that are of interest for monitoring bioremediation activity. The PCR product sizes are in a mass range that is accessible to analysis by MALDI-TOF mass spectrometry. A rapid purification procedure using commercially available reversed-phase cartridges was applied prior to MALDI-TOF analysis. A small aliquot (1.5%, 1.5 microL) from a single 100-microL PCR reaction was sufficient for reliable detection. No cross-amplification products were observed when primers designed for one bacterial species were used with genomic DNA of the other species. The methodology described here has potential to allow less expensive and faster characterization of the ability of microbial populations to destroy pollutants in groundwater and soil at contaminated industrial sites. 相似文献
7.
JS Lodmell JC Paillart D Mignot B Ehresmann C Ehresmann R Marquet 《Canadian Metallurgical Quarterly》1998,8(6):517-529
An essential step in the replication cycle of retroviruses is the dimerization of two copies of the genomic RNA. In vitro and in vivo studies have demonstrated that dimerization is mediated at least partially by RNA-RNA interactions. In HIV-1, the cis-element most important for dimerization is the dimerization initiation site (DIS), a stem-loop structure with an autocomplementary loop located between the primer binding site and the splice donor site in the 5' leader region of genomic RNA. We have studied the inhibition of dimerization of RNA corresponding to the first 615 nt of HIV-1 strains MAL and LAI in vitro using RNA and DNA oligonucleotides. The oligonucleotides were identical to or complementary to the DIS of the MAL and LAI strains, which are representative of the two most common DIS motifs found in natural isolates. The loop sequence of the DIS of the MAL isolate is AGGUGCACA, and that of the LAI sequence is AAGCGCGCA (the autocomplementary sequences are GUGCAC and GCGCGC, respectively). Several of the oligonucleotides were very efficient inhibitors of dimerization. However, homologous oligonucleotides displayed vastly different inhibition efficiencies between the two strains despite relatively modest sequence differences. Some of the oligonucleotides bound the viral RNA via a loop-loop interaction alone, whereas others recruited stem nucleotides to form an extended duplex even in the absence of loop complementarity. Furthermore, oligonucleotide inhibition was ineffective at low temperature, suggesting that a conformational change in the DIS is necessary for disruption of the dimeric structure of the DIS or binding of oligonucleotide or both. 相似文献
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RNA hairpins can alternatively form a dimer with a bulged loop flanked by regularly base paired regions. [1H]NMR spectroscopy and native gel electrophoresis were used to study how the sequence of nucleotides in the loop of the hairpin affect the hairpin-duplex interconversion. As a model system, a hairpin containing 7 nucleotides in the loop and 5 base pairs in the stem was used. The loop size was gradually reduced from 7 to 4 nucleotides, yielding finally the stable UNCG tetraloop. Single nucleotide mutations were performed to investigate the influence of the self-complementarity of the loop sequence on the dimerization. The results demonstrate that (1) the initial fraction of hairpin is determined by concentration of the oligonucleotide, the annealing procedure, and the relative stability of the loop, (2) the degree of self-complementarity of the loop sequence of the hairpin governs the dimerization kinetics, and (3) oligonucleotides complementary to the loop sequence decrease the dimerization rate. We propose a secondary structure-based model for the dimerization reaction of RNA hairpins in which the formation of intermolecular base pairs between self-complementary nucleotides of the loops represents the nucleation step. 相似文献
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J Ni T Liu A Kolbanovskiy J Krzeminski S Amin NE Geacintov 《Canadian Metallurgical Quarterly》1998,264(2):222-229
Site-specific carcinogen-modified oligonucleotides are often used in site-directed mutagenesis and other biological and biochemical studies of structure-function relationships. Postsynthetic analysis and confirmation of the sites of carcinogen binding in such oligonucleotides is an important step in the characterization of these site-specific carcinogen-DNA adducts. It is shown here that negative ion mode electrospray tandem mass spectrometry methods and collision-induced dissociation offer a rapid and convenient approach for the sequencing of products derived from the reaction of the carcinogenic and mutagenic metabolite of benzo[a]pyrene, the diol epoxide r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), with the 11-mer oligonucleotide d(CATGCGGCCTAC). The site of reaction of anti-BPDE with either one of the three dG residues in this oligonucleotide can be accurately established by comparing the mass/charge ratios of the observed collision-induced dissociation fragments with calculated values. 相似文献
10.
Physico-chemical and immunological studies have been done in order to further characterize the distorsions induced in DNA by the interstrand cross-links formed between the antitumor drug cis-diamminedichloroplatinum (II) (cis-DDP) and two guanines on the opposite strands of DNA at the d(GC/GC) sites. Bending (45 degrees) and unwinding (79 +/- 4 degrees) were determined from the electrophoretic mobility of multimers of 21- 24-base pairs double-stranded oligonucleotides containing an interstrand cross-link in the central sequence d(TGCT/AGCA). The distorsions induced by the interstrand cross-link in the three 22-base pairs oligonucleotides d(TGCT/AGCA), d(AGCT/AGCT) and d(CGCT/AGCG) were compared by means of gel electrophoresis, circular dichroism, phenanthroline-copper footprinting and antibodies specifically directed against cis-DDP interstrand cross-links. The four different technical approaches indicate that the distorsions are independent of the chemical nature of the base pairs adjacent to the interstrand cross-link. The general conclusion is that the interstrand cross-link induces a bending and in particular an unwinding larger than other platinum adducts and the distorsions are independent of the nature of the bases (purine or pyrimidine) adjacent to the d(GC/GC) site. 相似文献
11.
Several studies have shown that ionizing radiation generates a wide spectrum of lesions to DNA including base modifications, abasic sites, strand breaks, crosslinks and tandem base damage. One example of tandem base damage induced by @OH radical inX-irradiated DNA oligomers is N -(2-deoxy-beta-d- erythro -pentofuranosyl)-formylamine/8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). In order to investigate the biological significance of such a tandem lesion, both 8-oxo-7,8-dihydroguanine and formylamine were introduced into synthetic oligonucleotides at vicinal positions using the solid phase phosphoramidite method. For this purpose, a new convenient method of synthesis of 8-oxodGuo was developed. The purity and integrity of the modified synthetic DNA fragments were assessed using different complementary techniques including HPLC, polyacrylamide gel electrophoresis, electrospray and MALDI-TOF mass spectrometry. The piperidine test applied to the double modified base-containing oligonucleotides revealed the high alkaline lability of formylamine in DNA. In addition, various enzymatic experiments aimed at determining biochemical features of such multiply damaged sites were carried out using the synthetic substrates. The pro-cessing of the vicinal lesions by nuclease P1, snake venom phosphodiesterase, calf spleen phospho-diesterase and repair enzymes including Escherichia coli endonuclease (endo) III and Fapy-glycosylase was studied and is reported. 相似文献
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Ultraviolet circular dichroism spectra are reported for the oligonucleotide d(A15G15) in aqueous solutions containing 5 mM MgCl2 at several temperatures and in the presence of partially complementary oligonucleotides. Oligonucleotides with several consecutive terminal guanine residues self-associate to form aggregates, called frayed wires, that consist of integer numbers of strands. A "stem" is formed through interactions between the guanine residues of the associated oligonucleotides, whereas the adenine "arms" remain single stranded. Upon subtracting the circular dichroism spectrum of d(A15) from that of d(A15G15), one obtains a spectrum that closely resembles previously published spectra of poly(G). Subtracting spectra measured at temperatures between 10 degrees C and 60 degrees C reveals the resultant spectra to be independent of temperature, consistent with the extreme thermal stability observed for the aggregated structures. Upon the addition of d(T15) to the solution, complexes with the adenine portion of the d(A15G15) frayed wires are formed. Subtraction of d(A15):d(T15) spectra measured at several temperatures from those of the d(A15G15):d(T15) does not significantly alter the spectrum of the guanines. The helix-coil transition temperature of d(A15):d(T15) duplex is identical to that of the unbinding of d(T15) from d(A15G15):d(T15) complexes. Experiments using oligonucleotides in which the adenines were replaced with sequences of bases yielded similar results. By varying the length of the nonguanine tract, it is shown that the solubility of the complexes increases with the length of the nonguanine region of the oligonucleotide. 相似文献
14.
P Herdewijn 《Canadian Metallurgical Quarterly》1996,58(4):359-381
Among the approaches which have been followed to convert a natural antisense phosphodiester oligonucleotide into a potential therapeutic agent, conjugation chemistry seems to be one of the most attractive. Indeed, natural phosphodiester oligonucleotide have the ideal properties (sequence specific hybridization, RNaseH activation, low or no toxicity, water solubility, easy and relative inexpensive synthesis in bulk quantities) to function as antisense oligomers. Their disadvantages are situated in their nuclease lability so that they are rapidly degraded in a biological medium, and to their low cellular uptake due to their polyanionic character. We investigated the minimum molecular modifications necessary to transform natural, partial self-complementary, phosphodiester oligonucleotides into a nuclease stable construct which is taken up in sufficient amounts in tumor cells to exert a selective antiproliferative effect. This study revealed that small aliphatic diols connected at the 3'-end gives oligonucleotides which are stable against nuclease degradation and which demonstrate potent and selective biological activity. Because of the low toxicity of both phosphodiester oligonucleotides and most aliphatic diols no cytotoxicity and no side effects are expected for these constructs. Moreover, these oligonucleotides may be synthesized easily in large amounts for an affordable price. As a first potential application we demonstrate that a 1,3-propanediol modified 12-mer directed at the point-mutation in codon 12 of the Ha-ras mRNA demonstrates a selective antiproliferative effect at a concentration which is 500 times lower than the one observed with unmodified antisense oligonucleotides. The EC50 value of +/-nM warrants further development of these constructs as antitumoral drugs for these cancers showing a high frequency of Ha-ras oncogene expression with point mutation at codon 12. 相似文献
15.
I Pontén JM Sayer AS Pilcher H Yagi S Kumar DM Jerina A Dipple 《Canadian Metallurgical Quarterly》1999,38(3):1144-1152
Diastereomeric N6-substituted dAdo adducts (cis B[c]PhDE-2/1R and cis B[c]PhDE-2/1S) that correspond to cis-opening at C-1 of the enantiomeric benzo[c]phenanthrene 3,4-diol 1,2-epoxides in which the epoxide oxygen and the benzylic hydroxyl group are trans (DE-2) were synthetically incorporated into oligonucleotide 16-mers. Each adduct was placed at the fourth nucleotide from the 5'-end of each of two different oligonucleotide sequences derived from the E. coli supF gene. Each adduct was also placed in two additional oligonucleotide sequences that were constructed by interchanging the adduct site and the immediately adjacent nucleotides between the two original sequences. These oligonucleotides were designed for use in site-specific mutation studies, with a single-stranded bacteriophage M13mp7L2 vector, to determine if the effects of sequence context on types and frequencies of base substitution mutations are attributable only to nucleotides immediately adjacent to these polycyclic aromatic hydrocarbon diol epoxide-dAdo adducts, or whether more distant nucleotide residues also affect the mutagenic response. In SOS-induced Escherichia coli SMH77, total base substitution mutation frequencies for the cis B[c]PhDE-2/1R-dAdo adduct were relatively low (0.62-5.6%) compared with those for the cis B[c]PhDE-2/1S-dAdo adduct (11.9-56.5%). Depending on sequence context, cis B[c]PhDE-2/1R-dAdo gave predominantly A-->T or a more equal distribution of A-->T and A-->G mutations whereas cis B[c]PhDE-2/1S-dAdo gave either predominantly A-->T or predominantly A-->G base substitutions. Our results clearly indicate that nucleotides that are distal as well as those that are proximal to the adduct site are capable of influencing both the mutation frequency and the distribution of base substitution mutations. 相似文献
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Synthesis of the oligonucleotides conjugated with amino derivatives of beta-cyclodextrin and adamantane, at the 3'-end of host oligonucleotide, has been described. The oligonucleotide conjugates were examined for their nuclease stability, hybridization properties, and cellular uptake. The oligonucleotide conjugates had increased nuclease resistance compared to their parent oligonucleotides. Conjugation of adamantane to the oligonucleotides did not adversely affect the ability of the oligonucleotides to hybridize with their complementary RNA. Conjugation with amino derivatives of beta-cyclodextrin, however, significantly destabilized the duplex formation. In the cellular uptake studies, we found that amino derivatives of beta-cyclodextrin attached at 3'-end of the oligonucleotides did not help to increase the uptake by cells. Cellular uptake of oligonucleotide-adamantane conjugates in association with 2-(hydroxypropyl)-beta-cyclodextrin (HPCD) as a "carrier" was significantly higher than that of control oligonucleotides. 相似文献
17.
JG Scott N Liu Z Wen FF Smith S Kasai CE Horak 《Canadian Metallurgical Quarterly》1999,226(2):347-353
Hepatitis C virus (HCV) is the major cause of non-A, non-B hepatitis worldwide. Current treatments are not curative for most infected individuals, and there is an urgent need for both novel therapeutic agents and small-animal models which can be used to evaluate candidate drugs. A small-animal model of HCV gene expression was developed with recombinant vaccinia virus vectors. VHCV-IRES (internal ribosome entry site) is a recombinant vaccinia viral vector containing the HCV 5' nontranslated region (5'-NTR) and a portion of the HCV core coding region fused to the firefly luciferase gene. Intraperitoneal injection of VHCV-IRES produced high levels of luciferase activity in the livers of BALB/c mice. Antisense oligonucleotides complementary to the HCV 5'-NTR and translation initiation codon regions were then evaluated for their effects on the expression of these target HCV sequences in BALB/c mice infected with the vaccinia virus vector. Treatment of VHCV-IRES-infected mice with 20-base phosphorothioate oligonucleotides complementary to the sequence surrounding the HCV initiation codon (nucleotides 330 to 349) specifically reduced luciferase expression in the livers in a dose-dependent manner. Inhibition of HCV reporter gene expression in this small-animal model suggests that antisense oligonucleotides may provide a novel therapy for treatment of chronic HCV infection. 相似文献
18.
To better understand the uptake of oligonucleotides into cells, we have studied the labeling of cell surface proteins by an oligonucleotide conjugated to a radiolabeled photoactivatable crosslinker (Denny-Jaffe reagent). When HL60 cells are treated with the conjugate for 2 hours in a medium containing bovine serum albumin (BSA), almost all of the cell-associated label is found in one protein, which we identify as BSA. Cells grown and treated in a serum-free medium do not show this protein, whereas it is plainly seen in cells that are grown in serum-containing medium but then treated in serum-free medium. Overall association of the oligonucleotide with cells is much higher in serum-free medium than in BSA-containing medium, but the oligonucleotide is mostly not protein-associated in the absence of BSA. We conclude that (1) BSA from the medium serves to block overall association of oligonucleotide with cells, and (2) BSA is the main cell surface protein binding oligonucleotides. We discuss the possible role of albumin in endocytic uptake of oligonucleotides in the cell and in the biodistribution of oligonucleotides in vivo. 相似文献
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H Uchiyama K Hirano M Kashiwasake-Jibu B Mullah A Andrus K Taira 《Canadian Metallurgical Quarterly》1995,(34):111-112
A method was investigated for monitoring the integrity of oligonucleotides in solution and in cells using fluorescence resonance energy transfer (FRET) between two different fluorochromes attached to a single oligonucleotide. A 10-meric oligodeoxynucleotide labeled with fluorescein at its 5'-end and with rhodamine X at its 3'-end (F-ODN-R) was used. The oligomer had a specific absorption spectrum with peaks at 497 nm and 586 nm, which corresponded to fluorescein and rhodamine X, respectively. When excited at 494 nm, F-ODN-R had a specific fluorescence spectrum with peaks at 523 nm and 610 nm. The digestion of F-ODN-R with an endonuclease caused the increase in light intensity at 523 nm and the decrease at 610 nm. To examine effects in vivo, living sea urchin eggs were injected with a solution of F-ODN-R and excited with blue light at 470-490 nm. Two fluorescent images, a green image at 520-560 nm and a red image at above 580 nm, were obtained when a single egg was viewed under a fluorescence microscope. Eggs injected with the digested F-ODN-R emitted only green fluorescence. These results indicated that the integrity of oligonucleotides can be estimated in living cells by monitoring FRET after double-labeling of the oligonucleotides with fluorescein and rhodamine X. 相似文献
20.
Based on the oligomer-chip technology, oligonucleotide arrays were synthesized directly on polypropylene sheets by a modified phosphoramidite chemistry using beta-eliminating nucleobase-protecting groups in combination with a succinate solid-phase linker. This method decouples the oligonucleotide deprotection from the support cleavage procedure, in contrast to standard phosphoramidite chemistry. In addition to being reliable substrates for hybridization experiments, the arrays also serve as source for the isolation of individual oligonucleotides. Technically, this allowed for a direct control of the quality of the arrayed oligomers. The released compounds were sufficient in amount and purity to work without further purification in PCR and DNA-sequencing reactions, with the results being identical to controls with commercially obtained primer molecules. Consequences for oligomer-chip hybridization procedures, the applicability of such hybrid-function arrays in, for example, diagnostics or comparative biology, and developments toward parallel primer synthesis are discussed. 相似文献