Nucleic Acids Research, 1995, Vol. 23, No. 12 2223-2228
© 1995
RNA |
Ribozyme-mediated RNA degradation in nuclei suspension
The Scripps Research Institute, Departments of Neuropharmacology and of Molecular and Experimental Medicine 10666 North Torrey Pines Road, La Jolla, CA 92037, USA 1Department of Molecular Biology and Gene Therapy, Loma Linda Medical Center Loma Linda, CA 92350, USA 2Max-Planck-lnstitut fur Experimentelle Medizin Hermann-Rein-Straße 3, 37075 Göttingen, Germany
* To whom correspondence should be addressed
Received February 15, 1995. Revised May 5, 1995. Accepted May 5, 1995.
Ribozymes containing 2'-fluoro- and 2'-amlno-modifled pyrimidine nucleosldes in combination with terminal phosphorothioate linkages were targeted against HTLV-I tax RNA. In oder to examine the activity of such chemically modified ribozymes in the nuclear environment, they were incubated with nuclei of a Tax-transformed mouse fibroblast cell line. Ribozyme cleavage of tax RNA was analyzed by the RNase protection assay. Comparison of the cleavage of tax RNA Isolated nuclei with that of tax RNA present in nuclei suspension revealed a 30 times more efficient cleavage of the latter one. Pre-treatment with protelnase K and SDS abolished the enhancement of the ribozyme-mediated RNA cleavage. Catalytically inactive ribozymes did not yield any cleavage products. These results demonstrate an augmenting effect of nuclear proteins on the ribozyme-mediated RNA cleavage.
+present address: MicroProbe Corporation, 1725 220th Street S. E., Bothell, Washington 98021
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