Jacqueline R. Wyatt

Predicting oligonucleotide affinity to nucleic acid targets

A computer program, OligoWalk, is reported that predicts the equilibrium affinity of complementary DNA or RNA oligonucleotides to an RNA target. This program considers the predicted stability of the oligonucleotide-target helix and the competition with predicted secondary structure of both the target and the oligonucleotide. Both unimolecular and bimolecular oligonucleotide self structure are considered with a user-defined concentration. The application of OligoWalk is illustrated with three comparisons to experimental results drawn from the literature.

Induction of endogenous Bcl-xS through the control of Bcl-x pre-mRNA splicing by antisense oligonucleotides.

Resistance to apoptosis, which plays an important role in tumors that are refractory to chemotherapy, is regulated by the ratio of antiapoptotic to proapoptotic proteins. By manipulating levels of these proteins, cells can become sensitized to undergo apoptosis in response to chemotherapeutic agents. Alternative splicing of the bcl-x gene gives rise to two proteins with antagonistic functions: Bcl-xL, a well-characterized antiapoptotic protein, and Bcl-xS, a proapoptotic protein. We show here that altering the ratio of Bcl-xL to Bcl-xS in the cell using an antisense oligonucleotide permitted cells to be sensitized to undergo apoptosis in response to ultraviolet B radiation and chemotherapeutic drug treatment. These results demonstrate the ability of a chemically modified oligonucleotide to alter splice site selection in an endogenous gene and illustrate a powerful tool to regulate cell survival.

Analgesic profile of intrathecal P2X3 antisense oligonucleotide treatment in chronic inflammatory and neuropathic pain states in rats

&NA; Extracellular adenosine triphosphate (ATP), acting at P2X ionotropic receptors, is implicated in numerous sensory processes. Exogenous ATP has been shown to be algogenic in both animals and humans. Research focus has been directed towards the P2X3 receptor, as it is preferentially expressed on nociceptive C‐fibers and its implication in pain processing is supported by an altered nociceptive phenotype in P2X3 knock‐out mice. In order to further characterize the role of P2X3 receptor activation in nociception, we evaluated the effects of continuous intrathecal administration of P2X3 antisense oligonucleotides for 7 days in the rat. P2X3 receptor antisense oligonucleotide treatment significantly decreased nociceptive behaviors observed after injection of complete Freunds adjuvant (CFA), formalin or &agr;&bgr;‐methylene ATP into the rats hind paw. The anti‐hyperalgesic effects of the antisense treatment in the CFA model of inflammatory pain were dose related. Similar effects were observed with two distinct P2X3 antisense oligonucleotides. These behavioral effects were significantly correlated with a decrease in P2X3 receptor protein expression in the dorsal root ganglia (DRG). In contrast, a decrease in P2X3 receptor protein expression in the DRG did not affect nociceptive behavior in the carrageenan model of acute thermal hyperalgesia. P2X3 receptor antisense oligonucleotide treatment also significantly reduced mechanical allodynia observed after spinal nerve ligation. Overall, the present data demonstrate that activation of P2X3 receptors contribute to the expression of chronic inflammatory and neuropathic pain states and that relief form these forms of chronic pain might be achieved by selective blockade of P2X3 receptor expression or activation.

Novel Guanosine Quartet Structure Binds to the HIV Envelope and Inhibits Envelope Mediated Cell Fusion

Abstract We have identified a phosphorothioate oligonucleotide T2G4T2, which is a potent inhibitor of HIV infection in vitro. The compound was identified by combinatorial screening of a library of all possible octanucleotide sequences. The oligonucleotide forms a parallel-stranded, tetrameric guanosine-quartet (G-quartet) structure which specifically binds to the HIV envelope glycoprotein (gp 120) and inhibits both cell-to-cell and virus-to-cell infection at submicromolar concentrations. In the current study we demonstrate that the tetramer inhibits the infection of laboratory-derived isolate of HIV-1 and HIV-2 in a variety of phenotypically distinct established human cell lines and a panel of biologically diverse clinical isolates in fresh human peripheral blood lymphocytes and macrophages. The compound was also active against all drug-resistant virus isolates tested. In combination with AZT, ISIS 5320 exhibits additive to slightly synergistic anti-HIV activity. Cell-based mechanism of action studies dem...