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Nucleic Acids Research, 1989, Vol. 17, No. 22 9193-9204
© 1989

MOLECULAR BIOLOGY

RNase H cleavage of RNA hybridized to oligonucleotides containing methylphosphonate, phosphorothioate and phosphodiester bonds

Paul J. Furdon, Zbigniew Dominski and Ryszard Kole*

Department of Pharmacology and Lineberger Cancer Research Center, University of North Carolina Chapel Hill, NC 27599, USA

*To whom correspondence should be addressed at University of North Carolina, Lineberger Cancer Research Center, CS #7295 Chapel Hill, NC 27599, USA

Received July 28, 1989. Revised October 6, 1989. Accepted October 6, 1989.

Three types of 14-mer oligonucleolides were hybridized to human ß-globin pre-mRNA and the resultant duplexes were tested for susceptibility to cleavage by RNase H from E.coli or from HeLa cell nuclear extract. The oligonucleotides contained normal deoxynucleotides, phosphorothioate analogs alternating with normal deoxynucleotides, or one to six methylphosphonate deoxynucleosides. Duplexes formed with deoxyoligonucleotides or phosphorothioate analogs were susceptible to cleavage by RNase H from both sources, whereas a duplex formed with an oligonucleotide containing six methylphosphonate deoxynucleosides alternating with normal deoxynucleotides was resistant. Susceptibility to cleavage by RNase H increased parallel to a reduction in the number of methylphosphonate residues in the oligonucleotide.

Stability of the oligonucleotides in the nuclear extract from HeLa cells was also tested. Whereas deoxyoligonucleotides were rapidly degraded, oligonucleotides containing alternating methylphosphortate residues remained unchanged after 70 minutes of incubation. Other oligonucleotides exhibited intermediate stability.


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