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.

effectively inhibited MS-2 RNA-directed protein synthesis but they had much less effect on either poly(U) or poly(A)-directed polypeptide synthesis. In the reticulocyte system, these compounds had no significant effect on the translation of globin mRNA. The observation that d[(ApGpGp,) 3HjT)J binds to 70S ribosomes (association constant, 2.0 x 10 M-, 37C) together with the specificity of the inhibitory action of these compounds on protein synthesis strongly suggests that inhibition of translation is a consequence of analogue binding to Shine-Dalgarno sequence of 16S rRNA. The oligonucleoside methylphosphonates inhibited both protein synthesis (without concurrent inhibition of RNA synthesis) and colony formation by E coli ML 308-225 (a permeable mutant) whose cell wall contains negligible quantities of lipopolysaccharide but had no effect on wild-type E. coli B. Our preliminary results on the uptake of oligodeoxyribonucleoside methylphosphonates by E. coli B show that these cells are not permeable to oligomers longer than 4 nucleotidyl units. Although oligodeoxyribonucleoside methylphosphonates are taken up by mammalian cells in culture, this series of analogues had negligible inhibitory effects on colony formation by transformed human cells. This study indicates that this class of nonionic oligonucleotide analogues can be used to probe and regulate the finction and structure of nucleic acids of defined sequence within living cells.Single-stranded exposed regions of cellular nucleic acids are potential target regions for base-pairing interactions with complementary oligonucleotides. Binding of oligonucleotides to these regions can be used to probe and regulate the structure-function relationship of nucleic acids in both biochemical and cellular systems. Deoxyribooligonucleotides complementary to the reiterated 3'-and 5'-terminal nucleotides of Rous sarcoma virus 35S RNA inhibited the translation of the RNA in a cell-free system as well as the virus production of chicken fibroblast tissue cultures (1, 2). Studies in our laboratory have shown that an oligonucleotide ethylphosphotriester complementary to the amino acid-accepting stem of most tRNAs had a transient but specific inhibitory effect on the growth of mammalian cells in culture (3). More recently, we have studied the effects of oligo(dA) methylphosphonate analogues (complementary to the anticodon loop of tRNAIVs) on bacterial and mammalian cells in culture (4). These analogues contain an isosteric 3'-5' linked methylphosphonate group which replaces the normal phosphodiester linkage of nucleic acids.

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Oligothymidylate analogues having stereoregular, alternating methylphosphonate/phosphodiester backbones, d-Tp(TpTp)4T isomers I and II and d-Tp(TpTp)3T(pT)1-5 isomers I and II, were prepared by methods analogous to the phosphotriester synthetic technique. The designations isomer I nd isomer II refer to the configuration of the methylphosphonate linkage, which is the same through each isomer. Analogues with the type I methylphosphonate configuration form very stable duplexes with poly(dA) while those with the type II configuration form either 2T:1A triplexes or 1T:1A duplexes with poly(dA) of considerably lower stabilities. The oligothymidylate analogues were tested for their ability to initiate polymerizations catalyzed by Escherichia coli DNA polymerase I or calf thymus DNA polymerase alpha on a poly(dA) template. Neither d-Tp(TpTp)4T nor d-Tp(T]Tp)3TpT served as initiators of polymerization while d-Tp(TpTp)3T(pT)2-5 showed increasing priming ability as the length of the 3'-oligothymidylate tail increased. Analogues with type I methylphosphonate configuration were more effective initiators than the type II analogues at 37 degrees C. The apparent activation energies of polymerizations initiated by d-Tp(TpTp)3T-(pT)4 and 5 isomer I were greater than those for reactions initiated by isomer II or d-(Tp)11T. The results suggest that DNA polymerase interacts with the charged phosphodiester groups of the primer molecule and may help stabilize primer/template interaction. At least two contiguous phosphodiester groups are required at the 3' end of the analogue primers in order for polymerization to occur. Interactions between the polymerase and primer also appear to occur with phosphodiester groups located at sites remote from the 3'-OH polymerization site and may be influenced by the configuration of the methylphosphonate group.