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Nucleic Acids Research, 1995, Vol. 23, No. 6 954-961
© 1995

MOLECULAR BIOLOGY

Single base discrimination for ribonuclease H-dependent antisense effects within intact human leukaemia cells

Richard V. Giles*, Carolyn J. Ruddell, David G. Spiller, John A. Green1 and David M. Tidd

Departments of Biochemistry PO Box 147, Liverpool L69 3BX, UK 1Medicine, University of Liverpool PO Box 147, Liverpool L69 3BX, UK

*To whom correspondence should be addressed

Received December 8, 1994. Revised January 31, 1995. Accepted January 31, 1995.

We have previously demonstrated, In vitro, that phosphodiester and phosphorothioate antisense oligodeoxynucleotides could direct ribonuclease H to cleave non-target RNA sites and that chimeric methyl-phosphonodiester/phosphodiester analogue structures were substantially more specific. In this report we show that such chimeric molecules can promote point mutation-specific scission of target mRNA by both Escherlchla coll and human RNases H In vitro. Intact human leukaemia cells ‘biochemically micro-Injected’ with antisense effectors demonstrated efficient suppression of target mRNA expression, it was noted that the chimeric methylphosphonodiester/ phosphodiester structures showed single base discrimination, whereas neither the phosphodiester nor phosphorothioate compounds were as stringent. Finally, we show that the antisense effects obtained in intact cells were due to endogenous RNase H activity.


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