Open AccessNumber and distribution of methyiphosphonate linkages in oligodeoxynucleotides affect exo- and endonuclease sensitivity and ability to form RNase H substrates
Author(s) -
Robin S. Quartin,
Christine L. Brakel,
G. Wetmur
Publication year - 1989
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/17.18.7253
Subject(s) - phosphodiester bond , nuclease , rnase h , biology , rna , rnase p , oligonucleotide , endonuclease , exonuclease , dna , deoxyribonuclease i , biochemistry , ribonuclease , cleavage (geology) , microbiology and biotechnology , duplex (building) , gene , polymerase , base sequence , paleontology , fracture (geology)
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.