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PubMed2006

Elaboration of silica colloid/polymer hybrid support for oligonucleotide synthesis.

One way to increase the sensitivity of DNA diagnostic assays developed on microarrays is to improve the solid phase that allows the extraction of the target from a biological sample, before detection. Two parameters are influencing the performances of this capture step: (i) the specific surface area being offered for the capture and (ii) the number and the accessibility of oligonucleotide probes immobilized on the surface. In this context, we have developed an attractive approach which fulfills these two points. Our strategy was to elaborate a new material of high specific surface area, suitable to serve as support for both solid-phase oligonucleotide synthesis and in vitro diagnostic assay. This material has consisted of aggregates of colloidal amino-silica nanoparticles covalently linked by poly(ethylene oxide) (PEO) arms. The aggregation of amino-silica particles in the presence of reactive bis-isocyanate PEO was achieved in a controlled manner. The aggregate size and structure were examined by microscopy. The specific surface area of this material was measured by nitrogen adsorption technique. The composition of aggregate was studied by thermogravimetry and X-ray photoelectron spectroscopy. Then, this material has been successfully used as support for oligonucleotide synthesis of high yield and purity. The resulting system will be further evaluated in a diagnostic assay on a microarray.

Adsorption↗
PubMed1995

Simple repetitive DNA sequences from primates: compilation and analysis.

Simple repeats composed of tandemly repeated units 1-6 nucleotides (nt) long have been extracted from a selected set of primate genomic DNA sequences. Of the 501 theoretically possible, different types of repeats only 67 were present in the analyzed database in at least two different size ranges over 12 nt. They include all simple repeats known to be polymorphic in the primate genome. A list of moderately expanding and nonexpanding oligonucleotide patterns has also been included. Furthermore, we have compiled statistical data with emphasis on the overall variability of the most abundant 67 types of repeats. We have demonstrated that the expandability of at least some simple repeats may be affected by the overall base composition and by flanking sequences. In particular, the occurrence of tandemly repeated CAG and GCC triplets in exons positively correlates with their G+C content. We also noted that in the vicinity of Alu sequences tetrameric repeats are more abundant than in the total genomic DNA. This paper can be used as a comprehensive guide in identification of the most abundant and potentially polymorphic simple repeats. It is also of broader significance as a step toward understanding the contribution of flanking sequences and the overall sequence composition to variability of simple repeats.

Animals↗
PubMed1999

Morphological and compositional changes in a planktonic bacterial community in response to enhanced protozoan grazing.

We analyzed changes in bacterioplankton morphology and composition during enhanced protozoan grazing by image analysis and fluorescent in situ hybridization with group-specific rRNA-targeted oligonucleotide probes. Enclosure experiments were conducted in a small, fishless freshwater pond which was dominated by the cladoceran Daphnia magna. The removal of metazooplankton enhanced protozoan grazing pressure and triggered a microbial succession from fast-growing small bacteria to larger grazing-resistant morphotypes. These were mainly different types of filamentous bacteria which correlated in biomass with the population development of heterotrophic nanoflagellates (HNF). Small bacterial rods and cocci, which showed increased proportion after removal of Daphnia and doubling times of 6 to 11 h, belonged nearly exclusively to the beta subdivision of the class Proteobacteria and the Cytophaga-Flavobacterium cluster. The majority of this newly produced bacterial biomass was rapidly consumed by HNF. In contrast, the proportion of bacteria belonging to the gamma and alpha subdivisions of the Proteobacteria increased throughout the experiment. The alpha subdivision consisted mainly of rods that were 3 to 6 microm in length, which probably exceeded the size range of bacteria edible by protozoa. Initially, these organisms accounted for less than 1% of total bacteria, but after 72 h they became the predominant group of the bacterial assemblage. Other types of grazing-resistant, filamentous bacteria were also found within the beta subdivision of Proteobacteria and the Cytophaga-Flavobacterium cluster. We conclude that the predation regimen is a major structuring force for the bacterial community composition in this system. Protozoan grazing resulted in shifts of the morphological as well as the taxonomic composition of the bacterial assemblage. Grazing-resistant filamentous bacteria can develop within different phylogenetic groups of bacteria, and formerly underepresented taxa might become a dominant group when protozoan predation is the major selective pressure.

Animals↗
PubMed1984

Degrees of divergence in the E. coli genome from correlations between dinucleotide, trinucleotide and codon frequencies.

Oligonucleotide and codon frequencies have been determined in published sequences of E. coli DNA totaling 103,100bp with 18,459 reading frame trinucleotides; corresponding to 2.5% of the total genome. Dinucleotide frequencies are in excellent agreement with those determined by nearest neighbor chemical analysis, indicating the computer count of a limited sampling to be a good representation of the overall frequencies in total genomic DNA. The distinctive nonrandom codon pattern is found to be uniformly distributed and contributes to a distinctive nonrandom oligonucleotide pattern; enabling correlations between frequency levels to be extended beyond reading frame sequences. Correlation analysis indicates a surprisingly high degree of correlation everywhere in the genome. Coefficients of correlation between oligonucleotide frequencies overall and those in specific segments vary as follows: primary strands of individual coding sequences greater than 0.9 greater than lambda DNA greater than noncoding, non-RNA greater than phi X174 DNA greater than complementary strands greater than RNA genes congruent to 0.6 greater than transposon-insertion elements greater than T7DNA much greater than eukaryotic sequences congruent to 0. It is concluded that this high degree of oligonucleotide and codon correspondence in E. coli reflects the widespread distribution of remnants of an early and slowly changing codon pattern that has been continually dispersed by duplication-divergence processes, leading to the present genome.

Base Composition↗
PubMed1991

Complete nucleotide sequence of the Mycobacterium leprae 23 S and 5 S rRNA genes plus flanking regions and their potential in designing diagnostic oligonucleotide probes.

The complete nucleotide sequences of the Mycobacterium leprae 23 S and 5 S rRNA genes and their flanking regions are presented. As compared to other eubacterial homologous molecules the 23 S rDNA exhibits two insertions. A 16 nucleotide long insertion is almost unique to members of the genus Mycobacterium, while the second represents an extended version of helix 54. The potential of both insertions to serve as target for diagnostic oligonucleotide probes was proven by comparative sequence analysis of 23 S rRNA of several Mycobacterium species and by dot blot hybridization. In addition, a 19-mer oligonucleotide probe is described, which can be considered genus Mycobacterium-specific.

Base Composition↗
PubMed1993

Sequence-specific DNA damage using iodine-125-labeled antisense oligonucleotides.

A procedure is described that cleaves single-stranded DNA with sequence specificity. This process involves attaching a DNA damaging agent to an oligonucleotide. This oligonucleotide delivers the DNA damaging agent, iodine-125, to a specific DNA sequence by complementary hybridization. 5-[125I]Iodo-2-deoxycytidine 5'-triphosphate was enzymatically incorporated into an oligonucleotide that was designed to hybridize to a single-stranded DNA target. 125I decays by electron capture and causes breaks in the target DNA. These breaks were observed on a DNA sequencing gel. After 22 days of exposure to the 125I-labeled oligonucleotide, significant damage was observed within 1 to 2 bases of the expected site of hybridization. Densitometry showed that after 48 days the amount of damage had approximately doubled. This method facilitates easy design and testing of oligonucleotides that could potentially be used to inactivate gene expression in a wide variety of organisms.

Autoradiography↗
PubMed2005

A new approach to oligonucleotide N3'-->P5' phosphoramidate building blocks.

A new synthetic approach to 5-phosphoramidites of 3'-aminonucleosides was developed. The methodology relies upon the use of 3'-amino-2',3'-dideoxy nucleosides as the key starting materials. The final phosphoramidite products were obtained with high yields via 2-3-step efficient chemical transformations using selective introduction of orthogonal protective groups to the 3'-aminonucleoside sugar and base moieties.

Base Composition↗
PubMed1996

Identification of the template-binding cleft of T7 RNA polymerase as the site for promoter binding by photochemical cross-linking with psoralen.

We describe a novel method of photo-cross-linking DNA-binding proteins to DNA employing psoralen as a tether. We apply this method for the interaction of T7 RNA polymerase to its promoter. The crystallographic model of T7 RNA polymerase shows a cleft formed by the palm, thumb, and fingers domains. It was proposed that template DNA binds in the cleft. Here we directly and positively identify, in solution, the cleft as the seat of template binding. We photo-cross-linked a 23 bp promoter DNA to T7 RNA polymerase. We then determined the masses of cross-linked tryptic peptides by mass spectrometry and analyzed their amino acid composition. The cross-linked peptides were projected on the crystal structure of T7 RNA polymerase. The peptides nicely decorated the back, front, and side wall of the cleft. In a previous work [Sastry et al. (1993) Biochemistry 32, 5526-5538] we used site-specific psoralen furan-side monoadducts for cross-linking DNAs to DNA-binding proteins. We cross-linked a single-stranded 12-mer oligonucleotide to T7 RNA polymerase. We isolated and purified a DNA cross-linked tryptic peptide. We then used mass spectrometry and amino acid composition analysis to identify the location of this peptide on the T7 RNA polymerase primary sequence. In the present work we have mapped this peptide on the 3-D structure of T7 RNA polymerase. This peptide maps in the fingers domain of the polymerase. On the basis of a comparison of the map positions of peptides that cross-linked to either promoter DNA or single-stranded oligo-DNA, we propose that different functional domains may be involved in binding of double-stranded promoter DNA and nonspecific single-stranded DNA. Whereas the cleft of the polymerase is the seat of double-stranded promoter binding, the fingers domain may be used by the polymerase to grab single-stranded DNA (or RNA) in a nonspecific manner. Alternatively, the single-stranded oligo binding site may be an RNA product-binding site during transcription. The photochemical techniques we have developed [Sastry et al. (1993) Biochemistry 32, 5526-5538; this work] can be applied to other DNA-protein complexes to map DNA-binding domains.

Amino Acid Sequence↗
PubMed1997

Structure of oligonucleotides with either a strand break or a bulged nucleotide.

We report NMR and molecular modelling studies on a DNA duplex structure which is composed of three oligonucleotides and mimics a strand break. Although it retains a B form conformation our model suggests that it is kinked at the strand break. In the same sequence with an extra bulged adenosine at the centre for the major species this residue is stacked in the helix and a kink is observed in the model.

Base Composition↗
PubMed1988

Immunochemical detection of multiple conformations within a 36 base pair oligonucleotide.

A 36 base pair chimeric oligonucleotide containing a central core of DNA duplex flanked by RNA/DNA hybrid at each end was synthesized. These distinct regions of the oligonucleotide adopt different conformations which were detected with antibody probes. Enzyme linked immunosorbent assays (ELISA) and a gel electrophoresis retardation assay were used to demonstrate the binding of antibodies which recognize B-DNA, Z-DNA and RNA/DNA hybrid. The DNA duplex core of this oligonucleotide adopts the B-conformation in 0.14 M NaCl. In high salt solution (4 M NaCl) the DNA core adopts the Z-conformation. The RNA/DNA hybrid at the ends of the oligomer adopt a conformation which is distinct from both B-DNA and A-RNA.

Antibodies, Monoclonal↗
PubMed2004

DNA microarray analyses of the effects of dietary proteins.

Dietary proteins and amino acids serve not only as a building block for body components but also as regulators of a variety of body functions. A great many of the functions of ingested proteins, their peptide fragments, and amino acids have been characterized and some have been brought to practical application. Using a GeneChip DNA microarray system, we first compared the gene expression profiles among rats fed on 12% casein, 12% gluten, and protein-free diets for one week. The results revealed that a few hundred genes in the liver and muscle were up- or down-regulated by more than two-fold after feeding of the gluten or the protein-free diet. Interesting findings included the induction of genes for synthesis and catabolism of cholesterol by gluten feeding. In addition, we performed a study to examine the effect of the consumption of an enzymatically produced, hypoallergenic wheat flour on gene expression profiles in rats. The results confirmed the safety of this novel food product.

Animals↗
PubMed2006

Comparisons of PCR-based genome amplification systems using CpG island microarrays.

The characterization of complex DNA libraries by high-throughput sequencing technologies provides a powerful approach toward finding mutations and genetic variation in the human genome. However, the value of these analyses is highly dependent upon the quality of the DNA libraries themselves. For example, the sequence composition of libraries made using PCR-based procedures can be skewed due to biases in the amplification efficiency of individual library members. Here, we used CpG island microarrays to evaluate the biases incurred in PCR-based genome amplification using three different DNA polymerase mixtures designed to efficiently amplify problematic sequence tracts. Based on hybridization properties of dye-labeled DNA libraries to these microarrays, we quantified the overall and specific trends in the PCR efficiency of more than 1,400 sequences with high GC-content, which generally amplify with low efficiency using conventional PCR protocols. Overall, all three DNA polymerase mixtures produced libraries that show substantial increases in the representation of CpG island segments that poorly amplify with Taq DNA polymerase. However, the effects of these DNA polymerases were quite specific since they did not alter the relative representation of segments that efficiently amplify with Taq DNA polymerase. Furthermore, we demonstrate that DNA microarrays provide a robust platform for rapidly evaluating the ability of different PCR systems to amplify difficult genomic regions targeted for mutational, resequencing, and genotyping analyses.

Base Composition↗
PubMed1999

Effects of inulin on faecal bifidobacteria in human subjects.

A controlled study with eight healthy free-living subjects was carried out, in which energy intake was adjusted to the individual energy requirements. On administration of inulin, blood lipids, the faecal microflora, short-chain fatty acids and accompanying gastrointestinal symptoms were characterized in order to investigate the long-term effect of inulin. During the run-in phase (8 d), subjects received a typical Western diet providing 45% energy as fat and 40% energy as carbohydrate. Subsequently, the subjects consumed a fat-reduced diet which provided 30% energy as fat and 55% energy as carbohydrate for a period of 64 d using inulin as a fat replacer. The amounts of inulin consumed by the subjects (up to 34 g/d) were based on individual energy requirements with the aim to keep the diet isoenergetic with that used in the run-in period. To assess the effects of inulin administration, a control study (run-in and intervention) was carried out in which subjects consumed the same diet but devoid of inulin during the whole course of the study. To investigate the effect of inulin on faecal flora composition total bacteria and bifidobacteria in the faeces were enumerated by in situ hybridization with 16S rRNA targeted oligonucleotide probes. Inulin significantly increased bifidobacteria from 9.8 to 11.0 log10/g dry faeces and caused a moderate increase in gastrointestinal symptoms such as flatulence and bloatedness, whereas blood lipids and short-chain fatty acids remained essentially unaffected.

Adult↗
PubMed1988

Extra thymidine stacks into the d(CTGGTGCGG).d(CCGCCCAG) duplex. An NMR and model-building study.

NMR and model-building studies were carried out on the duplex d(CTGGTGCGG).d(CCGCCCAG), referred to as (9+8)-mer, which contains an unpaired thymidine residue. Resonances of the base and of several sugar protons of the (9+8)-mer were assigned by means of a NOESY experiment. Interresidue NOEs between dG(4) and dT(5) as well as between dT(5) and dG(6) provided evidence that the extra dT is stacked into the duplex. Thermodynamic analysis of the chemical shift vs temperature profiles yielded an average TmD value of 334 K and delta HD of -289 kJmol-1 for the duplex in equilibrium random-coil transition. The shapes of the shift profiles as well as the thermodynamic parameters obtained for the extra dT residue and its neighbours again indicate that the unpaired dT base is incorporated inside an otherwise intact duplex. This conclusion is further supported by (a) the observation of an imino-proton resonance of the unpaired dT; (b) the relatively small dispersion in 31P chemical shifts (approximately 0.5 ppm) for the (9+8)-mer, which indicates the absence of t/g or g/t combinations for the phosphate diester torsion angles alpha/zeta. An energy-minimized model of the (9+8)-mer, which fits the present collection of experimental data, is presented.

Base Composition↗
PubMed1998

Interdependence between DNA template secondary structure and priming efficiencies of short primers.

Here we analyze the effect of DNA folding on the performance of short primers and describe a simple technique for assessing hitherto uncertain values of thermodynamic parameters that determine the folding of single-stranded DNA into secondary structure. An 8mer with two degenerate positions is extended simultaneously at several complementary sites on a known template (M13mp18) using one, two or three (but never all four) of the possible dNTPs. The length of the extension is site specific because it is limited by the first occurrence in the downstream template sequence of a base whose complementary dNTP is not present. The relative priming efficiencies of different sites are then ranked by comparing their band brightnesses on a gel. The priming efficiency of a short primer (unlike conventional long primers) depends dramatically on the secondary structure of the template at and around the priming site. We calculated the secondary structure and its effect on priming using a simple model with relatively few parameters which were then optimized to achieve the best match between the predictions and the actual rankings of the sites in terms of priming efficiency. This work introduces an efficient and conceptually novel approach that in the future can make use of more data to optimize a larger set of DNA folding parameters in a more refined model. The model we used, however crude it may be, significantly improved the prediction of priming efficiencies of 8mer primers and appreciably raised the success rate of our DNA sequencing technique (from 67 to 91% with a significance of P < 7 x 10(-5)), which uses such primers.

Base Composition↗
PubMed2000

The spherulites(TM): a promising carrier for oligonucleotide delivery.

Concentric multilamellar microvesicles, named spherulites(TM), were evaluated as an oligonucleotide carrier. Up to 80% oligonucleotide was encapsulated in these vesicles. The study was carried out on two different spherulite(TM) formulations. The spherulite(TM) size and stability characteristics are presented. Delivery of encapsulated oligonucleotide was performed on a rat hepatocarcinoma and on a lymphoblastoid T cell line, both expressing the luciferase gene. We showed that spherulites(TM) were able to transfect both adherent and suspension cell lines and deliver the oligonucleotide to the nucleus. Moreover, 48-62% luciferase inhibition was obtained in the rat hepatocarcinoma cell line when the antisense oligonucleotide targeted to the luciferase coding region was encapsulated at 500 nM concentration in spherulites(TM) of different compositions.

Animals↗