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Hairpin ribozyme-antisense RNA constructs can act as molecular Lassos.

ABSTRACT: We have developed a novel class of antisense agents, RNA Lassos, which are capable of binding to and circularizing around complementary target RNAs. The RNA Lasso consists of a fixed sequence derived from the hairpin ribozyme and an antisense segment whose size and sequence can be varied to base pair with accessible sites in the target RNA. The ribozyme catalyzes self-processing of the 5'- and 3'-ends of a transcribed Lasso precursor and ligates the processed ends to produce a circular RNA. The circular and linear forms of the self-processed Lasso coexist in an equilibrium that is dependent on both the Lasso sequence and the solution conditions. Lassos form strong, noncovalent complexes with linear target RNAs and form true topological linkages with circular targets. Lasso complexes with linear RNA targets were detected by denaturing gel electrophoresis and were found to be more stable than ordinary RNA duplexes. We show that expression of a fusion mRNA consisting of a sequence from the murine tumor necrosis factor-alpha (TNF-alpha) gene linked to luciferase reporter can be specifically and efficiently blocked by an anti-TNF Lasso. We also show in cell culture experiments that Lassos directed against Fas pre-mRNA were able to induce a change in alternative splicing patterns.

SUBMITTER: Dallas A

PROVIDER: S-EPMC2588507 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Hairpin ribozyme-antisense RNA constructs can act as molecular Lassos.

Dallas Anne A Balatskaya Svetlana V SV Kuo Tai-Chih TC Ilves Heini H Vlassov Alexander V AV Kaspar Roger L RL Kisich Kevin O KO Kazakov Sergei A SA Johnston Brian H BH

Nucleic acids research 20081025 21

We have developed a novel class of antisense agents, RNA Lassos, which are capable of binding to and circularizing around complementary target RNAs. The RNA Lasso consists of a fixed sequence derived from the hairpin ribozyme and an antisense segment whose size and sequence can be varied to base pair with accessible sites in the target RNA. The ribozyme catalyzes self-processing of the 5'- and 3'-ends of a transcribed Lasso precursor and ligates the processed ends to produce a circular RNA. The ...[more]

PMID: 18953032

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Project description:BackgroundThe incidence of diabetic erectile dysfunction (ED) is rapidly increasing, and due to the severe angiopathy caused by diabetes, current drugs are ineffective at treating ED. Insulin-like growth factor-binding protein 5 (IGFBP5) promotes cell death and induces apoptosis in various cell types.ObjectivesTo evaluate the effectiveness of IGFBP5 knockdown in improving erectile function in diabetic mice.Materials and methodsDiabetes was induced by injecting streptozotocin (STZ) intraperitoneally into male 8-week-old C57BL/6 mice. Eight weeks after diabetes induction, mice were divided into four groups: a nondiabetic control group and three STZ-induced diabetic mice groups, which were administered intracavernous injections of phosphate buffered saline, scrambled control shRNA, or shRNA targeting mouse IGFBP5 (shIGFBP5) lentivirus particles. Two weeks later, we measured erectile function by electrically stimulating the bilateral cavernous nerve. To mimic diabetic angiopathy, primary cavernous endothelial cells (MCECs) from healthy mice were cultured and treated with glucose.ResultsIGFBP5 expression in MCECs or cavernous tissues were significantly increased under diabetic conditions, and knockdown of IGFBP5 induced MCECs angiogenic activity under high-glucose conditions. STZ-induced diabetic mice had reduced erectile function, but shIGFBP5 treatment resulted in significant improvements (to 90% of the nondiabetic control group level). Furthermore, in diabetic mice, numbers of cavernous endothelial cells, pericytes, and neuronal cells were increased by shIGFBP5 treatment, which also increased eNOS Ser1177 phosphorylation, decreased permeability and apoptosis of cavernous endothelial cells. In addition, IGFBP5 was found to mediate the AKT, ERK, p38 signaling pathways.Discussion and conclusionKnockdown of IGFBP5 improved erectile function in diabetic mice by promoting cell proliferation and reducing apoptosis and permeability. Local inhibition of IGFBP5 expression may provide a new treatment strategy for diabetic ED and other ischemic vascular or neurological diseases.

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Hairpin ribozyme-antisense RNA constructs can act as molecular Lassos.

Publications

Hairpin ribozyme-antisense RNA constructs can act as molecular Lassos.

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