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Nucleic Acids Research, 1993, Vol. 21, No. 18 4383-4391
© 1993

MOLECULAR BIOLOGY

Quantitative evaluation of intracellular sense: antisense RNA hybrid deuplexes

Sijian Wang and Bruce J. Dolnick*

Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute Elm and Carlton Streets, Buffalo, NY 14263, USA

*To whom correspondence should be addressed

Received March 12, 1993. Revised July 16, 1993. Accepted July 16, 1993.

Previous studies have demonstrated that for an antisense RNA to be effective in attenuating gene expression, a large but Indeterminate excess of antisense RNA is required. To quantitatively evaluate RNA hybrid duplex formation, expression vectors containing antisense dihydrofolate reductase (DHFR) cDNAs were transfected into KB and KB-1BT (a DHFR overexpresslng variant) cells and transfectants expressing antisense transcripts of exon 1 through intron I (ex1-l) or exons 1 through 4 (ex1-4) were analyzed for hybrid duplex formation. Stable duplexes were detectable in KB-1BT but not in KB cells. Approximately 5 - 9% of antisense ex1-l RNA and 20 - 37% of antisense ex1 - 4 RNA were found In duplexes. The amount of each hybrid duplex RNA was found to be a linear function of intracellular singlestranded antisense RNA levels and a hybrid index, Hs:ss was devised to describe this relationship. Based upon the value of Hs:ss for each antisense RNA:mRNA duplex, It is calculated that an approximate 2,800- and 600-fold excess of ex1-l and ex1 - 4 antisense RNA are respectively required for 50% of DHFR mRNA to be present in duplexes. Results support the hypothesis that Intracellular sense:antisense RNA hybrid duplex formation is inefficient and dependent upon the levels, lengths and possibly the structures of the RNAs involved.


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