Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.4 (RNase H)
 2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Scanning oligodeoxynucleotide (ODN) arrays appear promising in vitro tools for the prediction of effective antisense reagents but their usefulness has not yet been reported in mammalian systems. In this study, we have evaluated the use of scanning ODN arrays to predict efficacious antisense ODNs targeting the human epidermal growth factor receptor (EGFR) mRNA in a human epidermoid cancer cell line and in primary human glioma cells. Hybridisation accessibility profile of the first 120nt in the coding region of the human EGFR mRNA was determined by hybridising a radiolabelled EGFR transcript to a scanning array of 2684 antisense sequences ranging from monomers to 27-mers. Two ODNs, AS1 and AS2, complementary to accessible sequences within the EGFR mRNA, were designed and their ability to hybridise to EGFR mRNA was further confirmed by in vitro RNase H-mediated cleavage assays. Phosphorothioate-modified 21-mer AS1 and AS2 ODNs inhibited the growth of an established human A431 cancer cell line as well as primary glioma cells from human subjects when delivered as cationic lipoplexes. In contrast, scrambled controls and AS3-an antisense ODN complementary to an inaccessible site in EGFR mRNA-were inactive. Western blots showed that AS1 ODN exhibited a dose-dependent inhibition of EGFR protein expression in A431 cells in the nanomolar range. Microarray-based gene expression profiling studies of A431 cells treated with the 21-mer phosphorothioate AS1 ODN demonstrated successful inhibition of downstream signalling molecules further confirming the effective inhibition of EGFR expression in human cancer cells by antisense ODNs designed by scanning ODN array technology.
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PMID:Messenger RNA expression profiling of genes involved in epidermal growth factor receptor signalling in human cancer cells treated with scanning array-designed antisense oligonucleotides. 1294 63

The optimal design of hybridisation-competent antisense oligonucleotides (ODNs) coupled with an efficient delivery system appear to be important prerequisites for the successful use of antisense reagents for gene silencing. We selected an antisense ODN complementary to an accessible region of the epidermal growth factor receptor (EGFR) mRNA with the aid of an antisense oligonucleotide scanning array. The scanning array comprised 2684 antisense ODN sequences targeting the first 120 nts in the coding region of EGFR mRNA. The array-designed antisense ODN was covalently conjugated to a novel anionic dendrimer using a pentaerythritol-based phosphoroamidite synthon via automated DNA synthesis and the ability of this conjugate to effectively deliver and down-regulate EGFR expression in cancer cells was evaluated. Each dendrimeric structure had nine ODN molecules covalently linked to a common centre at their 3' termini. This dendrimer conjugate was markedly more stable to serum nucleases compared to the free ODNs and the cellular uptake of ODN-dendrimer conjugates was up to 100-fold greater as compared to mannitol, a marker for fluid phase endocytosis, and up to 4-fold greater than naked ODN in cancer cells. ODN-dendrimer uptake was energy-dependent and mediated, at least in part, via binding to cell surface proteins; a process that was inhibited by self-competition and by competition with free ODN, salmon sperm DNA, heparin and dextran sulphate. Fluorescent microscopy studies showed a combination of punctate and more diffuse cytosolic distribution pattern for fluorescently labelled ODN-dendrimer conjugate in A431 cells implying internalization by endocytosis followed by release and sequestration of the conjugate into the cytosol. Little or no conjugate appeared to be present in the nuclei of A431 cells. In vitro RNase H-mediated cleavage assays confirmed that covalently conjugated antisense ODNs in the dendrimer conjugate were able to hybridize and cleave the array-defined hybridisation target site within the EGFR mRNA without the need for ODN dissociation from the conjugate. In cell culture, ODN-dendrimer conjugates were effective in inhibiting cancer cell growth that correlated with a marked knockdown in EGFR protein expression. These data highlight a novel anionic dendrimer delivery system for gene silencing oligonucleotides that improved their biological stability, cellular delivery and antisense activity in cultured cancer cells.
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PMID:A novel anionic dendrimer for improved cellular delivery of antisense oligonucleotides. 1534 87