Biochemical and biological effects of nonionic nucleic acid methylphosphonates

Abstract

Oligodeoxyribonucleoside methylphosphonates with base sequences complementary to the anticodon loop of tRNALys and to the -ACCA-OH amino acid accepting stem of tRNA were prepared by chemical synthesis. Oligodeoxyadenosine methylphosphonates form stable, triple-stranded complexes with both poly(U) and poly(dT). These analogues selectively inhibit cell-free aminoacylation of tRNALys (E. coli) but have no effect on aminoacylation of tRNALys (rabbit). The extent of inhibition is temperature dependent and parallels the ability of the oligomer to bind to poly(U), which suggests that inhibition occurs as a result of oligomer binding to the -UUUU- anticodon loop of tRNALys (E. coli). The failure of the oligodeoxyadenosine methylphosphonates to inhibit tRNALys (rabbit) amino-acylation suggests that there may be a difference between the structure of tRNALys or its interaction with aminoacyl synthetase in the Escherichia coli and rabbit systems. The oligodeoxyadenosine analogues also effectively inhibit polyphenylalanine synthesis in cell-free translation systems derived from both E. coli and rabbit reticulocytes. The extent of inhibition parallels the Tm values of the oligo(A) phosphonate-poly(U) complexes and suggests that the inhibition is a consequence of complex formation with the poly(U) message. Tritium-labeled oligodeoxyribonucleoside methylphosphonates with a chain length of up to nine nucleotidyl units are taken up intact by mammalian cells in culture. All the oligomer analogues tested inhibited, to various extents, colony formation by bacterial, hamster, and human tumor cells in culture.

Partial protection of oncogene, anti-sense oligodeoxynucleotides against serum nuclease degradation using terminal methylphosphonate groups

“…However, we are presently continuing our work with chimeric methylphosphonodiester/phosphodiester oligodeoxynucleotides on the basis that by combining the desirable properties of both structures we may produce a superior anti-sense effector. In particular, by extending the methylphosphonate sequences at each end of the molecule we may enhance cell uptake of the oligodeoxynucleotide in addition to protecting the molecule from exonucleases (Miller et al, 1981). In addition, by reducing the length of the internal phosphodiester sequence we may reduce the target for endonucleolytic degradation while retaining the ability of the molecule to direct ribonuclease-H cleavage of mRNA at the site of hybridisation (Donis-Keller, 1979), an anti-sense mechanism of sequence specific protein synthesis inhibition not exhibited by homogeneous methylphosphonate oligonucleotide analogues (Maher & Dolnick, 1988).…”

Specific Inhibition of Influenza Virus RNA Polymerase and Nucleoprotein Gene Expression by Liposomally Encapsulated Antisense Phosphorothioate Oligonucleotides in MDCK Cells

“…Although viral replication could be inhibited by unmodified oligonucleotides, the problems of nuclease sensitivity and low cellular uptake limit in vivo applications (Hélène & Toulmé, 1990;Erickson & Yzant, 1992). Investigations have focused on designing more stable oligonucleotide derivatives, such as methylphosphonates (Miller et al, 1981), phosphorothioates (Stec et al, 1984) and phosphoramidites (Agrawal et al, 1988). Several reports have described the development of phosphorothioate oligonucleotides as potential antiviral therapeutic agents.…”

Site specificity of the inhibitory effects of oligo(nucleoside methylphosphonate)s complementary to the acceptor splice junction of herpes simplex virus type 1 immediate early mRNA 4.

“…The 5' terminal nucleoside is linked by a phosphodiester group, which increases the solubility of the oligomer and allows it to be phosphorylated by polynucleotide kinase (8). These analogues are resistant to nuclease hydrolysis and penetrate mammalian cells in culture (9). They specifically inhibit the expression of target genes (10,11).…”