Number and distribution of methylphosphonate linkages in oligodeoxynucleotides affect exo- and endonuclease sensitivity and ability to form RNase H substrates.
Oligodeoxynucleotides with different arrangements of methylphosphonate linkages were examined for nuclease sensitivity in vitro, stability in tissue culture, and ability to form RNase H-sensitive substrates with complementary RNA. After nuclease treatment, resistance was demonstrated by the ability to alter the electrophoretic mobility of a labeled complementary phosphodiester oligodeoxynucleotide. Both 5'- and 3'-exonuclease activities were retarded by methylphosphonate linkages. Methylphosphonate-containing oligodeoxynucleotides with 1-5 adjacent phosphodiester linkages were tested as substrates for the endonucleases DNase I and DNase II. The results indicated that a span of three or fewer contiguous internal phosphodiester linkages led to the greatest resistance to endonuclease. However, in serum-supplemented culture medium half-lives of these oligodeoxynucleotides were independent of the number of contiguous phosphodiester linkages. Methylphosphonate-containing oligodeoxynucleotides were hybridized to RNA runoff transcripts and tested as substrates for RNase H. The results indicated that a span of three internal phosphodiester linkages in the oligodeoxynucleotide was necessary and sufficient to direct cleavage of the RNA in the duplex.
Quartin
R S
RS
Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029.
Brakel
C L
CL
Wetmur
J G
JG
D001483:Base Sequence; D003470:Culture Media; D004720:Endonucleases / Q000378:metabolism*; D004722:Endoribonucleases / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides; D063065:Organophosphonates; D012313:RNA / Q000378:metabolism; D016914:Ribonuclease H; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D003470:Culture Media; D009838:Oligodeoxyribonucleotides; D063065:Organophosphonates; D012313:RNA; D004720:Endonucleases; D004722:Endoribonucleases; D005092:Exonucleases; D016914:Ribonuclease H