Methyl phosphonoamidites are deoxynucleoside amidites modified such that, when incorporated into an oligonucleotide, that base position will have a (electrically neutral) methyl phosphonate backbone linkage instead of the standard (negatively charged) phosphodiester linkage. Oligos containing one or more methyl phosphonate linkages will be resistant to nuclease degradation at those positions, and the lack of charge improves intracellular transport. Because of these properties, methyl phosphonolated oligos have been explored as anti-sense reagents (1). However, since methyl phosphonate linkages lower the oligo’s cellular uptake (2) as well as the Tm of the duplex formed with its RNA target (3), and, most importantly, also interferes with activation of RNase H activity (4), considerable care must taken in choosing which, and how many, methyl phosphonate linkages to incorporate into a putative anti-sense oligo. In that regard, we note that 2’-O-Methyl RNA oligos containing a single 3’-end methyl phosphonate “cap” (to eliminate 3’-exonuclease degradation) have been successfully used as anti-sense reagents (5). In addition, DNA extension primers containing such a "cap" have been used to characterize the nuclease activity of the yeast telomerase complex (6). Methylphosphonolated anti-sense oligos have also been used successfully to “mask” sites in U1 and U2 snRNPs required for spliceosome formation, and thus interfere with mRNA splicing (7). Many of the unique properties of methylphosphonolated oligos are due to the introduction of chirality into the phosphodiester backbone by the methyl group (8).
1. Sarin, P.S., Agrawal, S., Civeira, M.P., Goodchild, J., Ikeuchi, T., Zamecnik, P.C. Inhibition of acquired immunodeficiency syndrome virus by oligodeoxynucleoside methylphosphonates. Proc. Natl. Acad. Sci. USA (1988), 85: 7448-7451.
2. Blake, K.R., Murakami, A., Spitz, S.A., Glave, S.A., Reddy, M.P., Ts’o, P.O., Miller, P.S. Hybridization arrest of globin synthesis in rabbit reticulocyte lysates and cells by oligodeoxyribonucleoside methylphosphonates.Biochemistry (1985), 24: 6139-6145.
3. Kibler-Herzog, L., Zon, G., Uznanski, B., Whittier, G, Wilson, W.D. Duplex stabilities of phosphorothioate, methylphosphonate, and RNA analogs of two DNA 14-mers. Nucleic Acids Res. (1991), 19: 2979-2986.
4. Walder, J. Antisense DNA and RNA: progress and prospects. Genes Dev. (1988), 2: 502-504.
5. Prater, C.E., Miller, P.S. 3’-Methylphosphonate-Modified Oligo-2’-O-methylribonucleotides and Their Tat Peptide Conjugates: Uptake and Stability in Mouse Fibroblasts in Culture. Bioconjugate Chem. (2004), 15: 498-507.
6. Niu, H., Xia, J., Lue, N.F. Characterization of the Interaction between the Nuclease and Reverse Transcriptase Activity of the Yeast Telomerase Complex. Mol. Cell. Biol. (2000), 20: 6806-6815.
7. Temsamani, J., Agrawal, S., Pederson, T. Biotinylated Antisense Methylphosphonate Oligodeoxynucleotides—Inhibition of Spliceosome Assembly and Affinity Selection for U1 and U2 Small Nuclear RNPs. J. Biol. Chem. (1991), 266: 468-472.
8. Thiviyanathan, V., Vyazovkina, K.V., Gozansky, E.K., Bichenchova, E., Abramova, T.V., Luxon, B.A., Lebedev, A.V., Gorenstein, D.G. Structure of Hybrid Backbone Methylphosphonate DNA Heteroduplexes: Effect of R and S Stereochemistry. Biochemistry. (2002), 416: 827-838.
- Methyl Phosphonate dT. (mp)dT