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PubMed1989

High resolution chromatography of ribonucleosides and its application to RNA analysis.

A simple and precise method was developed for the separation of nucleosides including modified nucleosides and oligonucleotides. Nineteen kinds of nucleosides were completely separated by HPLC using an ODS column (TSK-gel ODS 80TM) and aqueous mobile phases. The RNA molecule was digested by base restrictive RNase (RNase A, RNase T1) and the digests were separated chromatographically into each oligonucleotide. The nucleoside composition of an oligonucleotide was then determined by this analytical system. It is thus possible to fit the oligonucleotide in the original RNA molecule by using modified bases as markers. The reaction site of quinacrine mustard for tRNA(Phe) (from yeast) could be determined by this analytical system.

Base Sequence↗
PubMedDate not supplied

[Interactions of gene 5 protein of bacteriophage f1 with oligonucleotides of a certain composition and sequence].

Interaction of gene 5 protein of bacteriophage f1 with a set of oligodeoxyribonucleotides (including those with 1,N6-ethenoadenine), which simulate a site of attachment of protein on DNA has been studied. The tyrosine and ethenoadenine fluorescence and CD of the complexes of the protein with the oligonucleotides have been measured. It has been revealed that the stoichiometry of the complexes depends on the protein--oligonucleotide ratio. It is demonstrated that protein binding changes the degree of hydration of the oligonucleotides and the geometry of stacking, without marked unstacking. Tyrosine residues partially intercalate in the oligonucleotide. It is possible that one of the tyrosines sits between the second and the third and one of the phenylalanines--between the third and the forth bases, of the pentanucleosidetetraphosphate from the 5'-end.

Base Sequence↗
PubMed1977

Use of an amino-silica column for the high-performance liquid chromatographic analysis of synthetic oligodeoxy-nucleotides.

The use of an amino-silica column in the chromatographic analysis of synthetic oligodeoxyribonucleotides and their derivatives from different stages of oligonucleotide synthesis has been investigated. By eluting with 0.10 M potassium phosphate solution of pH 3.30, the nucleotide composition of oligonucleotides can be established within 15 min. In a linear gradient of phosphate buffer (0.10-0.75 M) at neutral pH, the separation of oligonucleotides by length and in an acidic medium pH 3.30-4.30) by composition is possible; the oligonucleotides may be in the free form or modified by the various protecting groups used in synthetic oligonucleotide chemistry. The analysis of some reaction mixtures from different stages of oligonucleotide synthesis and of a number of synthetic oligodeoxyribonucleotides and their derivatives has been performed.

Amines↗
PubMed1996

An NMR self-diffusion study of the interactions between spermidine and oligonucleotides.

Self-diffusion coefficients have been determined by pulsed field gradient nmr methods for spermidine in solutions of the oligonucleotides d(GC)4 and d(GGAATTCC). The self-diffusion behavior of spermidine in solution of d(GC)4 is very similar to that observed previously for methylspermidine (completely N-methylated spermidine). Moreover, the self-diffusion behaviors of spermidine in solutions of d(GC)4 and d(GGAATTCC) are also quite similar, indicating that there is no significant influenceon on self-diffusion of oligonucleotide base composition. Furthermore, self-diffusion coefficients of the oligonucleotide d(GC)8 show only a small dependence on oligonucleotide concentration, and no measurable dependence on sodium ion or magnesium ion concentration.

Base Sequence↗
PubMed2026

Nucleotide Combination Proportions Across Algae, Monocotyledons and Dicotyledons: Insights into Plant Genome Evolution.

Plant evolution started with unicellular algae, gradually evolving multicellularity and terrestrial colonization. These evolutionary events were accompanied by the interplay of chromosome polyploidization, rearrangement, gene loss, and point mutation. We counted the proportion of nucleotide combinations in the genome sequences of 64 sequenced plants, and analyzed the significant difference in these nucleotide combination proportions among algae, monocotyledons and dicotyledons. The correlation of highly significant different and no significant different nucleotide combinations was analyzed respectively. Nucleotide combinations and their reverse complementary sequence proportions were analyzed in different functional regions of the genome. These results reveal that some nucleotide combinations are subject to strict selection, and these combinations have a higher proportion in the CDS regions and lower proportion in the intergenic regions. Meanwhile, there are some nucleotide combinations that are under less selective pressure, and these combinations have a higher proportion in the intergenic regions and lower proportion in the CDS regions. Cluster analysis based on trinucleotide to octanucleotide combination proportions reveals that plant genome evolution is accompanied by clade-wide differentiation of genome-wide nucleotide composition patterns, in addition to well-documented chromosomal polyploidization, structural rearrangement and gene loss events. We analyzed the changes in the proportion of nucleotide combinations at the genome level in 64 sequenced plants, providing a new idea for studying genome evolution in the plant kingdom.

comparative genomics↗
PubMed2006

Assembly of hybrid oligonucleotide modified gold (Au) and alloy nanoparticles building blocks.

The alloy-based hybrid materials with macroscopic network arrays were developed by AuAg/Au and AuAgPd/Au nanoparticle composites through oligonucleotides hybridization. AuAg/Au and AuAgPd/Au exhibited distinct organization. The morphology of AuAg/Au conjugation assembled mainly as compact aggregates while AuAgPd/Au hybrid conjugated into the loosen network assemblies. The dehybridization temperatures were studied as a function of molar ratio of alloy/Au. It was found that higher alloy/gold molar ratio led to stronger hybridization for alloy/gold composite, accompanied with increased melting temperature. These results could be interpreted in terms of more alloy nanoparticles bound to a Au particle when the molar ratio of alloy/gold increased. The thermal analysis also showed that AuAg/Au exhibited higher dehybridization temperature. A modified model describing the dehybridization probability of an intact Au/alloy aggregate was performed to support the dehybridization temperature increased with increasing alloy/Au molar ratio. As to more oligonucleotides carried by AuAg (4.9 +/- 1.9 nm) than by AuAgPd (4.4 +/- 1.5 nm) due to larger size in AuAg, the efficient hybridization could result in higher dehybridization temperature in AuAg/Au.

Alloys↗
PubMed1997

Oligonucleotides with conjugated dihydropyrroloindole tripeptides: base composition and backbone effects on hybridization.

The ability of conjugated minor groove binding (MGB) residues to stabilize nucleic acid duplexes was investigated by synthesis of oligonucleotides bearing a tethered dihydropyrroloindole tripeptide (CDPI3). Duplexes bearing one or more of these conjugated MGBs were varied by base composition (AT- or GC-rich oligonucleotides), backbone modifications (phosphodiester DNA, 2'-O-methyl phosphodiester RNA or phosphorothioate DNA) and site of attachment of the MGB moiety (5'- or 3'-end of either duplex strand). Melting temperatures of the duplexes were determined. The conjugated CDPI3 residue enhanced the stability of virtually all duplexes studied. The extent of stabilization was backbone and sequence dependent and reached a maximum value of 40-49 degrees C for d(pT)8. d(pA)8. Duplexes with a phosphorothioate DNA backbone responded similarly on CDPI3 conjugation, although they were less stable than analogous phosphodiesters. Modest stabilization was obtained for duplexes with a 2'-O-methyl RNA backbone. The conjugated CDPI3 residue stabilized GC-rich DNA duplexes, albeit to a lesser extent than for AT-rich duplexes of the same length.

Nucleic Acid Hybridization↗
PubMed2002

Can composition and structural features of oligonucleotides contribute to their wide-scale applicability as random PCR primers in mapping bacterial genome diversity?

Among current genotypic methodologies, random amplification of polymorphic DNA (RAPD or AP-PCR) represents a widely employed assay for the evaluation of bacterial genomic diversity. A common bottleneck of this technique, however, is represented by the screening of useful informative primers to discriminate among isolates of a particular bacterial species. In an attempt to simplify this process, we evaluated here the utility of degenerate oligonucleotides to act as informative AP-PCR primers. For this purpose, a number of features (G+C contents, degeneracy rate, modifications at the 5' end) of related degenerate primers was tested for their effects in the generation of informative arrays from a set of bacterial genomes. Our results indicate that a combination of a wide base composition and a common palindromic structure at the 5' end of the sequences that compose the degenerate primers tested here beneficially resulted for the generation of informative arrays aimed to evaluate the bacterial genome heterogeneity.

5' Flanking Region↗
PubMed1994

Capillary gel electrophoresis of oligonucleotides: prediction of migration times using base-specific migration coefficients.

Chemically synthesized oligodeoxyribonucleotides were subjected to capillary gel electrophoresis on three different polyacrylamide-based matrices. Analysis of about 1000 samples over a 1-year period showed that the gel matrix evolved with time resulting in shifting migration times, making it essential to use an internal standard. Cross-linked polyacrylamide matrices had the highest stability, allowing an average of 100 injections on the same capillary. Computer-aided prediction of migration times was subsequently evaluated to confirm the size and base composition of oligonucleotides more accurately. A number of problems were noted when using this approach on a routine basis, such as insufficient stability of the gel matrices, effects of secondary structure on migration and insufficient differences in migration times for oligonucleotides containing > 50 bases. Capillary gel electrophoresis at pH 3.5 in replaceable gels showed that migration was mainly dependent on the charge per base ratio resulting in separations of significantly altered selectivity which complemented analyses under the commonly used basic pH conditions.

Base Sequence↗
PubMed2004

Non-contact production of oligonucleotide microarrays using the highly integrated TopSpot nanoliter dispenser.

For the first time we report on the production of oligonucleotide microarrays using a highly parallel and highly integrated, pressure driven TopSpot nanoliter dispenser. The system enables non-contact printing of different media like oligonucleotides, DNA or protein solutions. We optimized the printing buffer needed for oligonucleotides microarrays production with respect to two major aspects: microfluidical optimum for droplet dispensing and biochemical coupling efficiency on different commercially available microarray slides. Coefficient of variations (CVs) of generated spot diameters were measured to be smaller than 1% within one single dispensing nozzle and smaller than 1.5% within all 24 parallel nozzles of the printhead for all printing buffers used. No carry-over and no cross-talk was found, in extensive experiments with oligonucleotides. Optimized printing buffer compositions and concentrations for oligonucleotide microarrays were found, as well as optimized coupling protocols. Furthermore, buffers and protocols were adapted to a host of different microarray slides used. With this system, prime critical points of microarray production are solved, leading to high quality high throughput microarray fabrication.

Nanotechnology↗
PubMed1998

Cationic oligonucleotides can mediate specific inhibition of gene expression in Xenopus oocytes.

Base-specific hydrogen bonding between an oligonucleotide and the purines in the major groove of a DNA duplex provide an approach to selective inhibition of gene expression. Oligonucleotide-mediated triplex formation in vivo may be enhanced by a number of different chemical modifications. We have previously described an in vitro analysis of triplex formation using oligonucleotides containing internucleoside phosphate linkages modified with the cation N , N -diethyl-ethylenediamine (DEED). When compared with unmodified oligonucleotides of identical base composition, DEED-modified oligonucleotides were better able to form DNA triplexes under conditions that approximate the pH, magnesium and potassium levels found in vivo . Here we report the ability of DEED-modified oligonucleotides to inhibit the expression of plasmid DNA injected into Xenopus oocytes. Inhibition is specific to plasmids containing a triplex formation target and sensitive to sequence alteration in the triplex forming target site. Inhibition of gene expression was nearly complete when oligonucleotide and plasmid were mixed together prior to injection. Inhibition was partial when oligonucleotide was injected first and not evident when plasmid was injected and allowed to form chromatin prior to oligonucleotide injection. Thus, access to DNA is a determining factor in effective triplex inhibition of gene expression.

Animals↗
PubMed1977

[Macromolecular biochemistry of normal and pathological white blood cells in man].

Leucocytes from normal donors and leukemia patients were isolated and lebelled in vitro with 32P-orthophosphate in order to compare labelling characteristics of nuclear high-molecular weight RNA, labelling characteristics, nucleotide compositions and oligonucleotide frequencies of ribosomal 28 S RNA. These studies revealed 1. structural microheterogeneity of 28 S RNA between the various leukemia cells studies without presenting a leukemia-specific structural marker, 2. an impaired production of ribosomal 28 S RNA from its nuclear precursor 45 S RNA in acute myeloblastic leukemia compared to PHA-stimulated normal lymphocytes. In the second part of this work, the influence of RNA from immunocompetent lymphocytes on the PHA-stimulation of M. Hodgkin lymphocytes was analyzed; the third part deals with studies on macromolecular carriers forcytostatic anthracyclines in human leukemia cells.

Hodgkin Disease↗
PubMed1998

Modified base compositions at degenerate positions of a mutagenic oligonucleotide enhance randomness in site-saturation mutagenesis.

Site-saturation mutagenesis, using degenerate oligonucleotide primers, is a frequently used method in introducing various mutations in a selected target codon. Oligonucleotides that are synthesized using equimolar concentrations of nucleoside phosphoramidites (dA, dC, dG, dT) in the positions to be saturated, result in a mutant population that is biased towards the original nucleotides. We found that this bias could be eliminated by modifying the concentrations of nucleoside phosphoramidites during the oligonucleotide synthesis. We synthesized eight degenerate oligonucleotides to saturate eight different codons, and sequenced a total of 344 mutagenized codons. In six of these eight oligonucleotides, we reduced to varying extents the concentrations of those nucleotides in the target positions that would form base pairs with the template. From the data, we analyzed the effects of different base compositions in the oligonucleotides when mutagenizing different codons, the influence of the positions of mismatches, and the significance of different non-Watson-Crick base pairs. Based on these results, we suggest levels to which different phosphoramidites should be reduced when synthesizing oligonucleotides for site-saturation mutagenesis.

Base Composition↗
PubMed1984

The 6S RNA transcribed from rodent total DNA in vitro is the transcript of the type 2 Alu family.

We have performed in vitro transcription, using total DNA isolated from mouse and a cloned mouse DNA fragment containing a representative type 2 Alu sequence characterized by Kominami et al. (1983) as templates. From each template, 6S RNA was transcribed. The fingerprint of the 6S RNA produced from in vitro transcription of total DNA showed a very clear pattern of oligonucleotides. By comparing the oligonucleotides of the 6S RNA produced from each template, we demonstrated that the 6S RNA transcribed from mouse total DNA is the in fact the transcript of type 2 Alu sequences. The composition of the oligonucleotides of the 6S RNA transcribed from mouse total DNA seems to reflect well that of the consensus sequence of the type 2 Alu family proposed by Krayev et al. (1982). In vitro transcripts of rat or hamster total DNA showed fingerprint patterns nearly identical to that of mouse DNA, confirming that rodents have equivalent type 2 Alu sequences whose transcripts have the same length and similar compositions of oligonucleotides. Thus total DNA transcription provides a novel approach to the detection and analysis of type 2 Alu sequences transcribed in vitro.

Animals↗
PubMed1997

Separation of oligonucleotides of identical size, but different base composition, by free zone capillary electrophoresis in strongly acidic, isoelectric buffers.

A novel method for analyzing oligonucleotides of the same length, but bearing a single base substitution, is reported, based on free zone capillary electrophoresis (CZE) under rather acidic pH values. For this purpose, a set of four 18-mers of fairly random base composition has been synthesized, bearing, in nucleotide 9, the following bases: T, C, G or A. Theoretical predictions, based on titration curves of single free nucleotides, allowed us to predict that the simultaneous separation of a mixture of all four oligonucleotides could be possible in a pH 3-4 window. In fact, electrophoresis at pH 5.7 gave a single, asymmetric peak, whereas CZE at pH 4.8 could resolve three out of four species (the T9 and G9 oligonucleotides co-migrating into a single zone). A unique separation power could be obtained at pH 3.3 in a buffer comprising an amphoteric species (isoelectric iminodiacetic acid, IDA) and 7 M urea. Although IDA exhibited a pI of 2.23 (for a 100 mM solution), the addition of 7 M urea (necessary to denature the oligonucleotides) raised the apparent pH of the solution to 3.3.

Acids↗
PubMed1998

Nuclease-resistant composite 2',5'-oligoadenylate-3', 5'-oligonucleotides for the targeted destruction of RNA: 2-5A-iso-antisense.

A new modification of 2-5A-antisense, 2-5A-iso-antisense, has been developed based on a reversal of the direction of the polarity of the antisense domain of a 2-5A-antisense composite nucleic acid. This modification was able to anneal with its target RNA as well as the parental 2-5A-antisense chimera. The 2-5A-iso-antisense oligonucleotide displayed enhanced resistance to degradation by 3'-exonuclease enzyme activity such as that represented by snake venom phosphodiesterase and by that found in human serum. 2-5A-Iso-antisense was able to effect the degradation of a synthetic nontargeted substrate, [5'-32P]pC11U2C7, and two targeted RNAs, PKR and BCR mRNAs, in a cell-free system containing purified recombinant human 2-5A-dependent RNase L. These results demonstrated that the novel structural modification represented by 2-5A-iso-antisense provided a stabilized biologically active formulation of the 2-5A-antisense strategy.

DNA, Complementary↗
PubMed1985

Base composition-independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries.

An oligonucleotide hybridization procedure has been developed that eliminates the preferential melting of A X T versus G X C base pairs, allowing the stringency of the hybridization to be controlled as a function of probe length only. This technique, which uses tetramethylammonium chloride, is especially helpful whenever a highly complex library is screened with a pool of oligonucleotide probes, which usually vary widely in base composition. The procedure can also be applied advantageously whenever an exact match to an oligonucleotide probe is desired, such as in screening for clones having as little as a single-base alteration generated by in vitro mutagenesis.

Base Composition↗