Signal amplification through nucleotide extension and excision on a dendritic DNA platform.

Techniques that provide strong signal amplification are useful in diagnostic applications, especially in detecting low concentrations of non-amplifiable target molecules. A versatile and strong signal amplification method based on activities of a DNA polymerase to generate high concentrations of pyrophosphate (PPi) is described. The generation of PPi is catalyzed by nucleotide extension and excision activities of a DNA polymerase on an oligonucleotide cassette. The signal is generated upon enzymatic conversion of PPi to ATP and ATP levels subsequently detected with firefly luciferase. Bioluminescence produced by an oligonucleotide cassette consisting of just two polymerase reaction sites is sufficient to detect them at low attomole levels. The attachment of a large number of these oligonucleotide cassettes to DNA dendrimers enabled the detection of such poly-valent substrate molecules at low zeptomole (10(-21)mol) concentrations. The extent of signal amplification obtained with dendrimer substrates is comparable to exponential target amplifications provided by nucleic acid amplification methods. The attachment of such PPi-generating dendritic DNA platforms to ligands that mediate target recognition would potentially permit detection of extremely low concentrations of analytes in diagnostic assays.

A novel, sensitive detection system for high-density microarrays using dendrimer technology.

To improve signal detection on cDNA microarrays, we adapted a fluorescent oligonucleotide dendrimeric signal amplification system to microarray technology. This signal detection method requires 16-fold less RNA for probe synthesis, does not depend on the incorporation of fluorescent dNTPs into a reverse transcription reaction, generates a high signal-to-background ratio, and can be used to allow for multichannel detection on a single chip. Furthermore, since the dendrimers can be detected individually, it may be possible, by employing dendrimer-binding standards, to calculate the numbers of bound cDNAs can be estimated. These features make the dendrimer signal detection reagent ideal for high-throughput functional genomics research.

Radiation-induced recombination is dependent on Ku80.

We have recently shown that irradiating cells prior to transfection induces recombination, as manifested by increased stable transduction of both plasmid and adenoviral vectors. We hypothesized that Ku proteins, which have previously been shown to be involved in both recombination and the repair of DNA damage after irradiation, would likely be important mediators of radiation-induced recombination. The present work demonstrates that Ku80 is essential for radiation-induced recombination. While human and hamster Ku80 are equally effective at restoring the transfection efficiency and radiation resistance of xrs-5 cells, human Ku80 is much more effective at radiation-induced recombination than hamster Ku80. This difference is not due to differences in Ku80 expression or DNA end-binding activity, but it may be due to structural differences between human and hamster Ku80.

Detection of Epstein-Barr virus EBER sequence in post-transplant lymphoma patients with DNA dendrimers.

The EBER RNAs are the most numerous viral transcripts in latently infected lymphocytes in healthy individuals and also in the tumor cells of Epstein Barr virus (EBV)-associated malignancies. A rise in EBV load in peripheral blood has been associated with the onset of post-transplant lymphoproliferative disease (PTLD) in immunocompromised patients. Treatment of PTLD with adoptive immunotherapy has made the rapid and accurate determination of EBV load essential. The relationship between EBV load and other EBV-associated malignancies, like Hodgkin's disease or AIDS-associated lymphoma, is unknown. In order to define viral load based on the number of EBV-infected cells in the peripheral blood, we developed a method which combines cellular dilution of peripheral blood mononuclear cells with the direct detection of EBER-1 RNA with DNA dendrimers. DNA dendrimers are large scaffolds of DNA which give at least a 500 1000-fold increase in detection of membrane bound nucleic acid over oilgonucleotide probes. The use of a novel class of these nucleic acid superstructures is described as a specific probe for EBER-1 detection. When two PTLD patients were analyzed for viral load with DNA dendrimers, at least one in 250000 peripheral blood mononuclear cells were shown to be infected with EBV.

Eukaryotic DNA does not form nucleosomes as readily as some prokaryotic DNA.

Nucleosomal-length DNA was prepared from the genomic DNA of various prokaryotic and eukaryotic organisms by limited nuclease digestion after reconstitution with core histones. The DNAs ranged in base composition from 26.5% to 72% guanosine-plus-cytosine (%GC). The nucleosomal-length DNAs were then used in a competitive reconstitution assay in order to quantitatively determine their relative abilities to form nucleosomes. The results of the assay indicate a linear dependence of the free energy of nucleosome formation on base composition and, surprisingly, show that several prokaryotic DNAs form nucleosomes as well as or better than eukaryotic DNAs.

Absence of a Ku-like DNA end binding activity in the xrs double-strand DNA repair-deficient mutant.

Double-strand DNA break repair is important in maintaining the genetic integrity of the genome. Using a mobility shift assay, we find that a protein, or complex of proteins, that is present in mammalian and yeast cells binds to the ends of double-strand DNA and renders the ends resistant to exonuclease digestion. Additionally, a mammalian DNA double-strand repair-deficient mutant, xrs, has no observable DNA end binding activity, while a revertant cell has wild-type activity. In addition, mobility supershift assays using monoclonal antibodies to the human Ku antigen (M(r) 70,000 subunit) reveal that one of the proteins of this end binding activity may be the Ku antigen or a protein with similar antigenic determinants. These observations suggest that this DNA end-binding protein may function in DNA repair.

Isolated oligopurine tracts do not significantly affect the binding of DNA to nucleosomes.

Nucleosomal-length DNA was constructed to contain one of two 10 bp oligopurine-oligopyrimidine sequences, either d(A10.T10) or d(G10.C10). The 146 base pair (bp) sequences were then each tandemly cloned. This allowed for the production of circularly-permuted sequence variants in which the oligopurine tract was located at eight different positions. The permuted sequences were then assayed for their ability to reconstitute into nucleosomes by competitive reconstitution. The results of the assay indicate that the free energy of nucleosome formation differs only by several tenths of a kilocalorie per mole for an oligopurine tract at any position along the DNA, including the central dyad region.