Sequence requirements of oligonucleotide chiral selectors for the capillary electrophoresis resolution of low-affinity DNA binders.

We recently reported that a great variety of DNA oligonucleotides (ONs) used as chiral selectors in partial-filling capillary electrophoresis (CE) exhibited interesting enantioresolution properties toward low-affinity DNA binders. Herein, the sequence prerequisites of ONs for the CE enantioseparation process were studied. First, the chiral resolution properties of a series of homopolymeric sequences (Poly-dT) of different lengths (from 5 to 60-mer) were investigated. It was shown that the size increase-dependent random coil-like conformation of Poly-dT favorably acted on the apparent selectivity and resolution. The base-unpairing state constituted also an important factor in the chiral resolution ability of ONs as the switch from the single-stranded to double-stranded structure was responsible for a significant decrease in the analyte selectivity range. Finally, the chemical diversity enhanced the enantioresolution ability of single-stranded ONs. The present work could lay the foundation for the design of performant ON chiral selectors for the CE separation of weak DNA binder enantiomers.

Separation and identification of oligonucleotides by hydrophilic interaction liquid chromatography (HILIC)-inductively coupled plasma mass spectrometry (ICPMS).

A method for the separation and detection of oligonucleotides utilizing hydrophilic interaction liquid chromatography (HILIC) with inductively coupled plasma mass spectrometry (ICPMS) is described. Polythymidylic acids of various lengths (10, 15, 20 and 30 nucleotides) were separated under gradient HILIC conditions. Selective detection of oligonucleotides was possible through monitoring m/z 47, corresponding to (31)P(16)O(+), using ICPMS. Oxygen was used as a reaction gas in the collision/reaction cell to produce PO(+) by reacting with phosphorus in the gas phase, thereby effectively eliminating the interferences for phosphorus normally seen at m/z 31. Limits of detections (LODs) were determined to be 1.69 pmol, 1.21 pmol, 1.0 pmol and 0.55 pmol loaded on column for the 10, 15, 20 and 30 mer, respectively.

Genome-scale identification of nucleosome positions in S. cerevisiae.

The positioning of nucleosomes along chromatin has been implicated in the regulation of gene expression in eukaryotic cells, because packaging DNA into nucleosomes affects sequence accessibility. We developed a tiled microarray approach to identify at high resolution the translational positions of 2278 nucleosomes over 482 kilobases of Saccharomyces cerevisiae DNA, including almost all of chromosome III and 223 additional regulatory regions. The majority of the nucleosomes identified were well-positioned. We found a stereotyped chromatin organization at Pol II promoters consisting of a nucleosome-free region approximately 200 base pairs upstream of the start codon flanked on both sides by positioned nucleosomes. The nucleosome-free sequences were evolutionarily conserved and were enriched in poly-deoxyadenosine or poly-deoxythymidine sequences. Most occupied transcription factor binding motifs were devoid of nucleosomes, strongly suggesting that nucleosome positioning is a global determinant of transcription factor access.

Distribution properties of polymononucleotide repeats in molluscan genomes.

A total of 635 DNA sequences from 35 species of mollusks were used as taxonomic support to investigate several distribution features of polymononucleotides in genomic regions of different functionality. We show that all polymononucleotide types in mollusks fit to expectations in exons but not in nonexonic regions, in agreement with a leading role of negative selection on expansions/contractions of transcription-linked poly-(A/T) repeats. The fit of all repeat length types to an exponential decay precludes the existence of a threshold size for replication slippage, a popular but unsatisfactorily explained concept in mutation models for single repeats. The genomic density of poly-(A/T) repeats is not correlated with the DNA content of species, suggesting that the differential density of repeats between species could be better explained by the species-specific performance of its repair mechanisms. This research allows a better understanding of the distribution patterns of single repeats in eukaryotes.

Evidence of systematic expressed sequence tag IMAGE clone cross-hybridization on cDNA microarrays.

We present evidence of a potentially serious source of error intrinsic to all spotted cDNA microarrays that use IMAGE clones of expressed sequence tags (ESTs). We found that a high proportion of these EST sequences contain 5'-end poly(dT) sequences that are remnants from the oligo(dT)-primed reverse transcription of polyadenylated mRNA templates used to generate EST cDNA for sequence clone libraries. Analysis of expression data from two single-dye cDNA microarray experiments showed that ESTs whose sequences contain repeats of consecutive 5'-end dT residues appeared to be strongly coexpressed, while expression data of all other sequences exhibited no such pattern. Our analysis suggests that expression data from sequences containing 5' poly(dT) tracts are more likely to be due to systematic cross-hybridization of these poly(dT) tracts than to true mRNA coexpression. This indicates that existing data generated by cDNA microarrays containing IMAGE clone ESTs should be filtered to remove expression data containing significant 5' poly(dT) tracts.

Voltammetric determination of underivatized oligonucleotides on graphite electrodes based on their oxidation products.

A new electrochemical method to determine underivatized oligonucleotides is developed. The electro-oxidation of the adenine moieties of adsorbed oligonucleotides at elevated potentials on pyrolytic graphite electrodes (PGE) in neutral or alkaline media gives rise to electroactive products strongly adsorbed on the electrode surface. The extent of the redox processes of these products, with formal potential close to 0 V (vs Ag /AgCl) at pH 10, correlates well with the amount of parent oligonucleotide. Various electrochemical techniques have been compared and applied to the detection of specific DNA sequences and synthetic homopolynucleotides. Detection limits of 2 and 10 ng for (dA)20 and a 21-mer sequence of HIV-1, respectively, have been achieved using sample volumes of 10 microL. Moreover, the adsorbed oxidized oligonucleotide shows electrocatalytic activity toward the oxidation of NADH. The capability of the new method to detect DNA hybridization is discussed.

Novel signal amplification technology with applications in DNA and protein detection systems.

A non-enzymatic approach to signal amplification has practical advantages over conventional target amplification methods. We have designed a simple, cost-efficient signal amplification system that can be used to enhance the detection of nucleic acids or protein. The signal amplification process requires initial capture of analyte by a specific probe, which, depending on the analyte, can be an oligomer or an antibody. Once the analyte is captured, amplification moieties are applied to significantly enhance the sensitivity of analyte detection. Nucleic acid amplification is typically greater than 1000-fold, increasing the sensitivity of target detection to less than 1 amol/100 microL. This amplification strategy presents a very flexible system with components that are easily altered to accommodate diverse assay requirements.

Thermal denaturation of T4 gene 32 protein: effects of zinc removal and substitution.

Gene 32 protein (g32P), the single-stranded (ss) DNA binding protein from bacteriophage T4, is a zinc metalloprotein. The intrinsic zinc is one of the factors required for the protein to bind cooperatively to a ssDNA lattice. We have used differential scanning calorimetry to determine how the thermodynamic parameters characterizing the denaturation of g32P are affected by removal or substitution of the intrinsic zinc. Over a wide concentration range (1-10 mg/mL), the native Zn(II) protein unfolds at a tm of 55 degrees C with an associated mean enthalpy change of 139 kcal mol-1. Under the same conditions, the metal-free apoprotein denatures over a relatively broader temperature range centered at 49 degrees C, with a mean enthalpy change of 84 kcal mol-1. Substitution of Zn(II) in g32P by either Cd(II) or Co(II) does not significantly change the enthalpy of denaturation but does affect the thermal stability of the protein. All metallo forms of g32P when bound to poly(dT) undergo highly cooperative denaturational transitions characterized by asymmetric differential scanning calorimetry peaks with increases in tm of 4-5 degrees C compared to the unliganded metalloprotein. Removal of the metal ion from g32P significantly reduces the cooperativity of binding to poly(dT) [Giedroc, D. P., Keating, K. M., Williams, K. R., & Coleman, J. E. (1987) Biochemistry 26, 5251-5259], and presumably as a consequence of this, apo-g32P shows no change in either the shape or the midpoint of the thermal transition on binding to poly(dT).(ABSTRACT TRUNCATED AT 250 WORDS)

Single-strand binding proteins from phage T4 and E. coli form higher order structures with poly(dT).

Complexes of poly(dT) with gene 32 protein from phage T4 or E. coli single-strand binding protein were digested by nuclease P1 from Penicillum citrinum. Protected fragments were analyzed by gel electrophoresis. In both cases, a series of bands was obtained corresponding to multiples of a repeat unit whose size was about 80 nucleotides. Such protected fragments could not be detected under the same experimental conditions when poly(dA) was used instead of poly(dT). The formation of nucleosome-like structures is discussed in relation to the higher affinity exhibited by single-strand binding proteins towards poly(dT).

Structure of polyadenylic acid in the ribonucleic acid of Saccharomyces cerevisiae.

Investigations of the structure of polyadenylic acid [poly(A)] in yeast have shown that there are two classes of poly(A) distinguished by size and kinetics of synthesis. Each class is found directly on the 3' end of messenger RNA. One class contains poly(A) molecules ranging from 60 to less than 20 nucleotides long. The longest molecules in this poly(A) class are the first to become labeled when cells are exposed to [3H]adenine. Label then appears in progressively smaller molecules. The second class of poly(A) is about 20 nucleotides long. The length homogeneity of this class and the presence in nuclear DNA of many copies of a polythymidylate sequence which is the same length suggests that this poly(A) is synthesized by transcription from DNA.

Positioning of the A . T-rich regions in rat mitochondrial DNA by electron microscopy and analysis of the hysteresis of denaturation.

Intramolecular heterogeneity in the base composition of rat mitochondrial DNA (mtDNA) was shown by a combination of an improved denaturation mapping technique using electron microscopy and analysis of high-resolution optical melting-renaturation profiles. Circular mtDNA starts to melt in one specific region and then forms loops in four other regions in random order. These five early melting regions are all located in one half of the molecule. The arrangement of the early melting regions in rat mtDNA bears a remarkable resemblance not only to those of mtDNAs from several species of Drosophila but also to those of several species of Drosophila but also to those of several plasmid DNAs and phage DNA.

Analysis of DNA structure by hydroxyapatite columns and ethidium bromide fluorescence techniques. A comparative study and effect on DNA binding.

Seven duplex DNA preparations have been structurally analyzed by hydroxyapatite column chromatography and an ethidium bromide fluorescence technique. Significant contamination of one preparation with single-stranded DNA was detected by hydroxyapatite column chromatography. Five of the other six preparations were found to contain significant single-stranded regions by the ethidium bromide fluorescence technique. Synthetic poly dAT was found to be duplex in structure. The presence of single-stranded regions considerably influenced DNA binding results in a radioimmunoassay.

Structural organization and processing of the genetic transcript in the cellular slime mold Dictyostelium discoideum.

The organization of the genome and the synthesis and processing of heterogeneous nuclear RNA (HNRNA) in the cellular slime mold Dictysotelium discoideum have been analyzed. Approximately 60-70% of the genome of Dictyostelium consists of interspersed reiterated and single-copy sequences. The interspersed reiterated sequences have an average length of 250-400 nucleotides. Approximately 50% of the reiterated DNA sequences consist of long noninterspersed sequences. The results of analyses of ynRNA synthesis and processing have been incorporated into a model. According to the model the primary genetic transcript of Dictyostelium is synthesized as a molecule that is 25% larger than mRNA. The bulk of the hnRNA is synthesized from a unit consisting of a short reiterated DNA sequence transcript at the 5' end of the molecule and a single-copy sequence of approximately 1,200 nucleotides in length. In the processing of the mRNA precursor, there appears to be a loss of the majority of the repetitive sequence at the 5' end. The genome contains interspersed sequences of poly (dT)25. These sequences, which appear to be at the 3' terminus of the transcription unit, are transcribed directly into the heterogenous nuclear RNA and are contained within the messenger RNA. During the processing of the heterogeneous nuclear RNA, a poly (A) sequence of approximately 125 nucleotides in length is added posttranscriptionally to the 3' end of the molecule.