RNase H cleavage of RNA hybridized to oligonucleotides containing methylphosphonate, phosphorothioate and phosphodiester bonds.
Three types of 14-mer oligonucleotides were hybridized to human beta-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 methylphosphonate residues remained unchanged after 70 minutes of incubation. Other oligonucleotides exhibited intermediate stability.
Furdon
P J
PJ
Department of Pharmacology, University of North Carolina, Chapel Hill 27599.
Dominski
Z
Z
Kole
R
R
D001483:Base Sequence; D002467:Cell Nucleus / Q000201:enzymology; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005914:Globins / Q000235:genetics; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D009943:Organophosphorus Compounds; D012322:RNA Precursors / Q000235:genetics; D016914:Ribonuclease H; D013379:Substrate Specificity; D013873:Thionucleotides
D009838:Oligodeoxyribonucleotides; D009943:Organophosphorus Compounds; D012322:RNA Precursors; D013873:Thionucleotides; C032627:methylphosphonic acid; D005914:Globins; D004722:Endoribonucleases; D016914:Ribonuclease H