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PubMed2001

A laminated, flex structure for electronic transport and hybridization of DNA.

We have developed the first prototypes of a three-dimensional, electrophoretically driven microlaboratory for the analysis of proteins and DNA. By selecting the appropriate spacing and geometrical configuration, oligonucleotides were transported, in a controlled, rapid fashion, by electrophoresis in free-space. Transport efficiencies over 2 mm distances exceeded 70%. Electrodes of similar design were combined with an electronically addressed DNA hybridization chip to form a fully electrophoretic microlaboratory. In this instance, gold-plated copper electrodes were patterned on a 2 mil thick polyimide substrate. This polyimide layer was stiffened with 20 mil of polyimide to provide support for flip-chip bonding of our standard 100-site Nanochip. This composite structure illustrated three-dimensional transport of target oligonucleotides, through a via in the polyimide, along a series of electrodes and onto the diagnostic chip. Upon reaching the diagnostic chip, electronic hybridization was performed for a competitive reverse dot blot assay. The electronic assay showed a specific to nonspecific ratio in excess of 20:1. These results suggested that this type of structure might be of practical consequence with the development of a microlaboratory for biowarfare applications.

Base Sequence↗
PubMed2004

Structural analysis of hepatitis C RNA genome using DNA microarrays.

Many studies have tried to identify specific nucleotide sequences in the quasispecies of hepatitis C virus (HCV) that determine resistance or sensitivity to interferon (IFN) therapy, unfortunately without conclusive results. Although viral proteins represent the most evident phenotype of the virus, genomic RNA sequences determine secondary and tertiary structures which are also part of the viral phenotype and can be involved in important biological roles. In this work, a method of RNA structure analysis has been developed based on the hybridization of labelled HCV transcripts to microarrays of complementary DNA oligonucleotides. Hybridizations were carried out at non-denaturing conditions, using appropriate temperature and buffer composition to allow binding to the immobilized probes of the RNA transcript without disturbing its secondary/tertiary structural motifs. Oligonucleotides printed onto the microarray covered the entire 5' non-coding region (5'NCR), the first three-quarters of the core region, the E2-NS2 junction and the first 400 nt of the NS3 region. We document the use of this methodology to analyse the structural degree of a large region of HCV genomic RNA in two genotypes associated with different responses to IFN treatment. The results reported here show different structural degree along the genome regions analysed, and differential hybridization patterns for distinct genotypes in NS2 and NS3 HCV regions.

Conserved Sequence↗
PubMed1997

Synthesis of oligonucleotides containing two putatively mutagenic DNA lesions: 5-hydroxy-2'-deoxyuridine and 5-hydroxy-2'-deoxycytidine.

Spontaneous oxidative DNA damage occurs as a consequence of aerobic metabolism, lipid peroxidation, immune responses, ionizing radiation, and some chemical oxidants. These processes yield a vast array of oxidized DNA bases and sugars. The existence of significant steady-state levels of oxidized DNA bases in the genome suggests that these lesions are not completely repaired on a biologically relevant time scale and thus may contribute to mutagenesis. In particular, studies have shown that the steady-state levels of 5-hydroxy-2'-deoxycytidine (dC5-OH) and its deamination product, 5-hydroxy-2'-deoxyuridine (dU5-OH), are similar to those found for 7,8-dihydro-8-oxoguanosine, a known highly mutagenic lesion formed by oxidation of guanosine. Structural and biological properties of dC5-OH and dU5-OH have been constrained by the lack of synthetic methodology for oligonucleotides containing these modified bases. A method is described here for the solid-phase synthesis of oligonucleotides containing dC5-OH and dU5-OH. Preparation of each of the required phosphoramidites involved the selective protection of the base 5-hydroxyl group over the deoxyribose 5'- and 3'-hydroxyl groups. The base composition and the incorporation of the adducts into synthetic heptanucleotides were confirmed after purification of the modified oligonucleotides by enzymatic digestion and HPLC analysis. Mass spectrometric analysis of the oligonucleotide products by electrospray MS and GC/MS further confirmed their composition. Most significantly, deamination of the dC5-OH oligomer to a putative dU5-OH product during solid-phase DNA synthesis or oligonucleotide deprotection was not detected by any analytical technique employed.

Chromatography, High Pressure Liquid↗
PubMed2002

Solid-phase PCR in microwells: effects of linker length and composition on tethering, hybridization, and extension.

During the solid-phase PCR (SP-PCR), DNA oligonucleotides complementary to a soluble template and immobilized on a surface are extended in situ. Although primarily used for pathogen detection, SP-PCR has the potential for much broader application, including disease diagnostics, genotyping, and expression studies. Current protocols for SP-PCR in microwells are suitable for enzymatic detection of immobilized products, but yields are generally insufficient for direct detection of products using conventional fluorescent probes. Here, we quantitatively measure the outcome of tethering, hybridization, and solid-phase extension, and examine the effect of composition and length of the spacer at the 5' end of tethered oligonucleotides. Our results indicate that steric hindrance primarily affects polymerase activity rather than the efficiency of hybridization between the template and the tethered oligonucleotide. SP-PCR yields are significantly higher for a five-unit hexaethyleneglycol (HEG) spacer than for the more commonly used 10-residue deoxythymidine spacer. The optimal 5' HEG spacer resulted in a 60-fold increase in extension efficiency relative to a previously reported value for SP-PCR on a glass surface. Thus, optimized spacers should allow direct quantification of SP-PCR products, providing a simple, quantitative, and cost effective means of sample analysis for a variety of applications.

Arabidopsis↗
PubMed1978

[Topography of rRNA in ribosomes. Effect of pancreatic RNAse on small ribosomal subunits].

The treatment of E. coli 30S ribosome subunits with pancreatic RNase under certain conditions resulted in the release of rRNA (about 15%). The subunit retained as a whole structure: sedimentation coefficient was unchanged and no protein release was observed. The released RNA is a set of oligonucleotides from 1 to 9 bases, except hepta- and octanenucleotides. Base composition of this RNA fraction is similar to 16S RNA, a slight increase of purines content being due to the specificity of nuclease. Analysis of isoplit content has revealed that a spliting of long oligonucleotides in stechiometric amount from 30S subunits takes place: one nonanucleotide, one hexanucleotide and two pentanucleotides.

Escherichia coli↗
PubMed2001

ARP, a peptide derived from the stress-associated acetylcholinesterase variant, has hematopoietic growth promoting activities.

BACKGROUND: Psychological stress induces rapid and long-lasting changes in blood cell composition, implying the existence of stress-induced factors that modulate hematopoiesis. Here we report the involvement of the stress-associated "readthrough" acetylcholinesterase (AChE-R) variant, and its 26 amino acid C-terminal domain (ARP) in hematopoietic stress responses. MATERIALS AND METHODS: We studied the effects of stress, cortisol, antisense oligonucleotides to AChE, and synthetic ARP on peripheral blood cell composition and clonogenic progenitor status in mice under normal and stress conditions, and on purified CD34 cells of human origin. We employed in situ hybridization and immunocytochemical staining to monitor gene expression, and 5-bromo-2-deoxyuridine (BrdU), primary liquid cultures, and clonogenic progenitor assays to correlate AChE-R and ARP with proliferation and differentiation of hematopoietic progenitors. RESULTS: We identified two putative glucocorticoid response elements in the human ACHE gene encoding AChE. In human CD34+ hematopoietic progenitor cells, cortisol elevated AChE-R mRNA levels and promoted hematopoietic expansion. In mice, a small peptide crossreacting with anti-ARP antiserum appeared in serum following forced swim stress. Ex vivo, ARP was more effective than cortisol and equally as effective as stem cell factor in promoting expansion and differentiation of early hematopoietic progenitor cells into myeloid and megakaryocyte lineages. CONCLUSIONS: Our findings attribute a role to AChE-R and ARP in hematopoietic homeostasis following stress, and suggest the use of ARP in clinical settings where ex vivo expansion of progenitor cells is required.

Acetylcholinesterase↗
PubMed1994

Phosphorothioate oligonucleotides inhibit the replication of lentiviruses and type D retroviruses, but not that of type C retroviruses.

Phosphorothioate analogs of oligodeoxynucleotides at a concentration of 2 microM protected Himalayan tahr cells from infection by caprine arthritis encephalitis virus (CAEV) and equine dermis cells from infection by equine infectious anemia virus (EIAV). The characteristics of this inhibition against these lentiviruses are similar to those previously described for the inhibition of HIV-1 in ATH8 cells [17]. Thus, the 28-mer homo-oligomer of cytidine [S-(dC)28] was at least as effective as three anti-sense sequences targeted to the LTR, gag, and env regions of CAEV. The effectiveness of homo-oligomers of equal length was in the order C >> A > T, and a random 28-copolymer with a composition of 2C:1G was as effective as S-(dC)28. Shorter oligonucleotides were less effective (28 > 14 > 5 mers) for all base compositions tested. While replication of a simian type D retrovirus was inhibited by S-(dC)28, this compound did not inhibit the cytopathogenicity of two type C retroviruses, amphotropic murine leukemia virus (MuLV), and baboon endogenous virus, when they were tested in the same cell lines used to support the replication of lentiviruses. Southern blot analysis of the high molecular weight DNA of drug-treated CAEV-infected cells showed that S-(dC)28 was acting at or before the reverse transcription step. Our present data and the earlier finding that S-(dC)28 is a potent in vitro inhibitor of the MuLV reverse transcriptase [15] suggest that S-(dC)28 is acting very early in the replication cycle of these lentiviruses. Since MuLV reverse transcriptase is inhibited in vitro, but its replication is not blocked in permissive cells, our data suggest that the phosphorothioate oligonucleotides are preventing virus attachment.

Animals↗
PubMed2003

Synergistic effects of epoxy- and amine-silanes on microarray DNA immobilization and hybridization.

Most microarray slides are manufactured or coated with a layer of poly(L-lysine) or with silanes with different chemical functional groups, for the attachment of nucleic acids on to their surfaces. The efficiency with which nucleic acids bind to these surfaces is not high, because they can be washed away, especially in the case of spotting oligonucleotides. In view of this, we have developed a method to increase the binding capacity and efficiency of hybridization of DNA on to derivatized glass surfaces. This makes use of the synergistic effect of two binding interactions between the nucleic acids and the coating chemicals on the surface of the glass slides. The enhanced binding allows the nucleic acids to be bound tightly and to survive stringency washes. When immobilized, DNA exhibits a higher propensity for hybridization on the surface than on slides with only one binding chemical. By varying the silane concentrations, we have shown that maximal DNA oligonucleotide binding on glass surfaces occurs when the percentage composition of both of the surface-coating chemicals falls to 0.2%, which is different from that on binding PCR products. This new mixture-combination approach for nucleic-acid binding allows signals from immobilization and hybridization to have higher signal-to-noise ratios than for other silane-coated methods.

Amines↗
PubMed2000

Analysis of the degradation of oligonucleotide strands during the freezing/thawing processes using MALDI-MS.

Synthetic oligonucleotide strands ranging from 5 to 25 units in length are commonly used as standards, probes, and templates in various bioanalytical applications. Until recently, their preparation, storage, and handling were regarded as unimportant, but this work provides valuable information to the contrary. The systematic degradation of oligonucleotide strands during sample preparation is investigated by repeatedly freezing/thawing short strands followed by matrix-assisted laser desorption ionization mass spectrometric (MALDI-MS) analysis. It is shown here that the longevity of an oligonucleotide strand is dependent on several factors including base composition, solution concentrations, and strand length as well as thawing conditions. Several trends in strand robustness were established. Our studies reveal that the robustness of strands is base-dependent: T-mer > A-mer > C-mer > G-mer. Likewise, an increase in the length of the strands increases the tendency of a sample to degrade. Another observation included that samples of mixed bases degrade according to structural conformations. All of these observations are attributed to the fact that the samples undergo degradation during sample/solvent isolation during freezing.

Base Sequence↗
PubMed1974

DNA sequence analysis: a general, simple and rapid method for sequencing large oligodeoxyribonucleotide fragments by mapping.

Several electrophoretic and chromatographic systems have been investigated and compared for sequence analysis of oligodeoxyribonucleotides. Three systems were found to be useful for the separation of a series of sequential degradation products resulting from a labeled oligonucleotide: (I) 2-D electrophoresisdagger; (II) 2-D PEI-cellulose; and (III) 2-D homochromatography. System (III) proved generally most informative regardless of base composition and sequence. Furthermore, only in this system will the omission of an oligonucleotide in a series of oligonucleotides be self-evident from the two-dimensional map. The sequence of up to fifteen nucleotides can be determined solely by the characteristic mobility shifts of its sequential degradation products distributed on the two-dimensional map. With this method, ten nucleotides from the double-stranded region adjacent to the left-hand 3'-terminus and seven from the right-hand 3'-terminus of bacteriophage lambda DNA have been sequenced. Similarly, nine nucleotides from the double-stranded region adjacent to the left-hand 3'-terminus and five nucleotides from the right-hand terminus of bacteriophage phi80 DNA have also been sequenced. The advantages and disadvantages of each separation system with respect to sequence analysis are discussed.

Base Sequence↗
PubMed2006

Microarray-based identification of antigenic variants of foot-and-mouth disease virus: a bioinformatics quality assessment.

BACKGROUND: The evolution of viral quasispecies can influence viral pathogenesis and the response to antiviral treatments. Mutant clouds in infected organisms represent the first stage in the genetic and antigenic diversification of RNA viruses, such as foot and mouth disease virus (FMDV), an important animal pathogen. Antigenic variants of FMDV have been classically diagnosed by immunological or RT-PCR-based methods. DNA microarrays are becoming increasingly useful for the analysis of gene expression and single nucleotide polymorphisms (SNPs). Recently, a FMDV microarray was described to detect simultaneously the seven FMDV serotypes. These results encourage the development of new oligonucleotide microarrays to probe the fine genetic and antigenic composition of FMDV for diagnosis, vaccine design, and to gain insight into the molecular epidemiology of this pathogen. RESULTS: A FMDV microarray was designed and optimized to detect SNPs at a major antigenic site of the virus. A screening of point mutants of the genomic region encoding antigenic site A of FMDV C-S8c1 was achieved. The hybridization pattern of a mutant includes specific positive and negative signals as well as crosshybridization signals, which are of different intensity depending on the thermodynamic stability of each probe-target pair. Moreover, an array bioinformatic classification method was developed to evaluate the hybridization signals. This statistical analysis shows that the procedure allows a very accurate classification per variant genome. CONCLUSION: A specific approach based on a microarray platform aimed at distinguishing point mutants within an important determinant of antigenicity and host cell tropism, namely the G-H loop of capsid protein VP1, was developed. The procedure is of general applicability as a test for specificity and discriminatory power of microarray-based diagnostic procedures using multiple oligonucleotide probes.

Animals↗
PubMed1999

Similarity in oligonucleotide usage in introns and intergenic regions contributes to long-range correlation in the Caenorhabditis elegans genome.

A method is presented which allows detection of a sequence correlation effect not related to patchiness in base composition or to preferences in codon usage. Recurrence plots providing local views of oligonucleotide recurrence regimen show that introns and intergenic regions are often characterised by a highly recurrent use of oligonucleotides. By window analysis it is possible to score a long sequence for the recurrence of a given subset of oligos while filtering away the effects of short-range correlations. Long-range exploration of chromosome III from Caenorhabditis elegans reveals that consistent use of recurrent oligonucleotides in introns and intergenic regions generates a correlation effect that extends over several megabases.

Animals↗
PubMed2005

Genome-wide selection of unique and valid oligonucleotides.

Functional genomics methods are used to investigate the huge amount of information contained in genomes. Numerous experimental methods rely on the use of oligo- or polynucleotides. Nucleotide strand hybridization forms the underlying principle for these methods. For all these techniques, the probes should be unique for analyzed genes. In addition to being unique for the studied genes, the probes should fulfill a large number of criteria to be usable and valid. The criteria include for example, avoidance of self-annealing, suitable melting temperature and nucleotide composition. We developed a method for searching unique and valid oligonucleotides or probes for genes so that there is not even a similar (approximate) occurrence in any other location of the whole genome. By using probe size 25, we analyzed 17 complete genomes representing a wide range of both prokaryotic and eukaryotic organisms. More than 92% of all the genes in the investigated genomes contained valid oligonucleotides. Extensive statistical tests were performed to characterize the properties of unique and valid oligonucleotides. Unique and valid oligonucleotides were relatively evenly distributed in genes except for the beginning and end, which were somewhat overrepresented. The flanking regions in eukaryotes were clearly underrepresented among suitable oligonucleotides. In addition to distributions within genes, the effects on codon and amino acid usage were also studied.

Amino Acids↗
PubMed1972

Distribution of pyrimidine oligonucleotides in strands L and H of Bacillus subtilis DNA.

The distribution of pyrimidine oligodeoxynucleotide clusters in L and H strands of Bacillus subtilis DNA separated by methylated albumin-Kieselguhr has been determined. Preparations of native and single-stranded DNA were degraded with diphenylamine in formic acid, and the released isostichs with the general formula of Py(n)P(n+1) were separated on DEAE-cellulose by chain length. Eleven isostichs were obtained for strands L and H in unequal proportions. Each isostich fraction was subfractionated by base composition on DEAE-cellulose at pH 3.0. 61 Pyrimidine oligonucleotide clusters were separated from the H strand and only 46 from the L strand. The findings show a higher degree of asymmetry between the strands in the distribution of cytosine-rich clusters as compared with thymine-rich clusters. The longest cytosine oligodeoxynucleotide present in both strands is of chain length 5. There is an unusually high distribution of thymine oligodeoxynucleotides of length 5-11. Up to chain length 6, the distribution of thymine oligodeoxynucleotides between the strands is about equal; from chain length 7 to 11 they occur predominantly in the H strand.

Amines↗
PubMed1997

Bipartite substrate discrimination by human nucleotide excision repair.

Mammalian nucleotide excision repair (NER) eliminates carcinogen-DNA adducts by double endonucleolytic cleavage and subsequent release of 24-32 nucleotide-long single-stranded fragments. Here we manipulated the deoxyribose-phosphate backbone of DNA to analyze the mechanism by which damaged strands are discriminated as substrates for dual incision. We found that human NER is completely inactive on DNA duplexes containing single C4'-modified backbone residues. However, the same C4' backbone variants, which by themselves do not perturb complementary hydrogen bonds, induced strong NER reactions when incorporated into short segments of mispaired bases. No oligonucleotide excision was detected when DNA contained abnormal base pairs without concomitant changes in deoxyribose-phosphate composition. Thus, neither C4' backbone lesions nor improper base pairing stimulated human NER, but the combination of these two substrate alterations constituted an extremely potent signal for double DNA incision. In summary, we used C4'-modified backbone residues as molecular tools to dissect DNA damage recognition by human NER into separate components and identified a bipartite discrimination mechanism that requires changes in DNA chemistry with concurrent disruption of Watson-Crick base pairing.

Base Composition↗
PubMed1997

Scavenger receptor-mediated delivery of antisense mini-exon phosphorothioate oligonucleotide to Leishmania-infected macrophages. Selective and efficient elimination of the parasite.

Targeted delivery of a 17-mer antisense phosphorothioate oligodeoxyribonucleotide, complementary to the common 5'-end of every mRNA of the parasite cells, to the phagolysosomes of cultured murine macrophages infected with Leishmania mexicana amazonensis selectively and efficiently eliminated the parasite cells without causing any detectable harm to the host cells. The antisense mini-exon oligonucleotide (ASM) was encapsulated into liposomes coated with maleylated bovine serum albumin (MBSA), the artificial ligand for macrophage scavenger receptors. MBSA-coating of the liposomes allowed specific binding of the liposomes to the macrophages, their receptor-mediated uptake, and subsequent degradation of the liposomes inside macrophage phagolysosomes to release ASM. When incubated with Leishmania-infected macrophages, MBSA-liposome-encapsulated ASM (10 microM) was able to kill >90% of the parasites within 5 hr as compared with 20% killing within this time period by free ASM. Oligonucleotides with complementary nucleotide sequence or with the same base composition as ASM but scrambled sequence had no antileishmanial effect under the conditions of the assay. This study reflects the efficacy of scavenger-receptor-mediated delivery of antisense phosphorothioate oligos in killing intraphagolysosomal pathogens.

Animals↗
PubMed2006

Abundant and diverse fungal microbiota in the murine intestine.

Enteric microbiota play a variety of roles in intestinal health and disease. While bacteria in the intestine have been broadly characterized, little is known about the abundance or diversity of enteric fungi. This study utilized a culture-independent method termed oligonucleotide fingerprinting of rRNA genes (OFRG) to describe the compositions of fungal and bacterial rRNA genes from small and large intestines (tissue and luminal contents) of restricted-flora and specific-pathogen-free mice. OFRG analysis identified rRNA genes from all four major fungal phyla: Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota. The largest assemblages of fungal rRNA sequences were related to the genera Acremonium, Monilinia, Fusarium, Cryptococcus/Filobasidium, Scleroderma, Catenomyces, Spizellomyces, Neocallimastix, Powellomyces, Entophlyctis, Mortierella, and Smittium and the order Mucorales. The majority of bacterial rRNA gene clones were affiliated with the taxa Bacteroidetes, Firmicutes, Acinetobacter, and Lactobacillus. Sequence-selective PCR analyses also detected several of these bacterial and fungal rRNA genes in the mouse chow. Fluorescence in situ hybridization analysis with a fungal small-subunit rRNA probe revealed morphologically diverse microorganisms resident in the mucus biofilm adjacent to the cecal and proximal colonic epithelium. Hybridizing organisms comprised about 2% of the DAPI (4',6-diamidino-2-phenylindole, dihydrochloride)-positive organisms in the mucus biofilm, but their abundance in fecal material may be much lower. These data indicate that diverse fungal taxa are present in the intestinal microbial community. Their abundance suggests that they may play significant roles in enteric microbial functions.

Animals↗
PubMed1997

A study of oligonucleotide occurrence distributions in DNA coding segments.

In this paper we present a general strategy designed to study the occurrence frequency distributions of oligonucleotides in DNA coding segments and to deal with the problem of detecting possible patterns of genomic compositional inhomogeneities and disuniformities. Identifying specific tendencies or peculiar deviations in the distributions of the effective occurrence frequencies of oligonucleotides, with respect to what can be a priori expected, is of the greatest importance in biology. Differences between expected and actual distributions may in fact suggest or confirm the existence of specific biological mechanisms related to them. Similarly, a marked deviation in the occurrence frequency of an oligonucleotide may suggest that it belongs to the class of so-called "DNA signal (target) sequences". The approach we have elaborated is innovative in various aspects. Firstly, the analysis of the genomic data is carried out in the light of the observation that the distribution of the four nucleotides along the coding regions of the genoma is biased by the existence of a well-defined "reading frame". Secondly, the "experimental" numbers found by counting the occurrences of the various oligonucleotide sequences are appropriately corrected for the many kinds of mistakes and redundancies present in the available genetic Data Bases. A methodologically significant further improvement of our approach over the existing searching strategies is represented by the fact that, in order to decide whether or not the (corrected) "experimental" value of the occurrence frequency of a given oligonucleotide is within statistical expectations, a measure of the strength of the selective pressure, having acted on it in the course of the evolution, is assigned to the sequence, in a way that takes into account both the value of the "experimental" occurrence frequency of the sequence and the magnitude of the probability that this number might be the result of statistical fluctuations. If the strength of the selective pressure evaluated in this fashion turns out to be sufficiently large, the corresponding sequence will be considered to have an occurrence frequency beyond expectations and, hence, to be statistically and biologically interesting.

Algorithms↗