Number and distribution of methyiphosphonate linkages in oligodeoxynucleotides affect exo- and endonuclease sensitivity and ability to form RNase H substrates

Abstract

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.

Oligonucleotides with novel, cationic backbone substituents: aminoethylphosphonates

“…One widely used approach to improve cell penetration involves removal of the negative charges to produce neutral backbones such as, for example, methyl phosphonates [1], phosphoramidates [2] or peptide nucleic acids [3]. Methylphosphonates have been shown to be transported into cells by endocytosis [1] and were more resistant to degradation by nucleases [4].…”

Morpholino antisense oligomer targeting human midkine: Its application for cancer therapy

“…Modifications that impart resistance to nuclease degradation include phosphorothioate (PS, Fig. 1c), 6,7 methylphosphonate, 8 and 2Ј-O-methyl-modified oligonucleotides. 9 PS-modified oligonucleotides are used commonly and effectively inhibit synthesis of proteins in various cell types.…”

Potential roles of antisense technology in cancer chemotherapy

“…In contrast, phosphorothioates are excellent substrates (Cazenave et al, 1989;Mirabelli et al, 1991;Stein and Cheng, 1993). In addition, chimeric molecules have been studied as oligonucleotides that bind to RNA and activate RNaseH (Furdon et al, 1989;Quartin et al, 1989). For example, oligonucleotides comprised of wings of 2'-methoxy phosphonates and a ®ve-base gap of oligodeoxynucleotides bind to their target RNA and activate RNaseH (Furdon et al, 1989;Quartin et al, 1989).…”

“…In addition, chimeric molecules have been studied as oligonucleotides that bind to RNA and activate RNaseH (Furdon et al, 1989;Quartin et al, 1989). For example, oligonucleotides comprised of wings of 2'-methoxy phosphonates and a ®ve-base gap of oligodeoxynucleotides bind to their target RNA and activate RNaseH (Furdon et al, 1989;Quartin et al, 1989). Furthermore, a single ribonucleotide in a sequence of deoxyribonucleotides was shown to be su cient to serve as a substrate for RNaseH when bound to its complementary deoxyoligonucleotide (Eder and Walder, 1991).…”