Selective inhibition of Escherichia coli protein synthesis and growth by nonionic oligonucleotides complementary to the 3′ end of 16S rRNA

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Abstract
A series of nonionic oligonucleotide analogs, the deoxyribooligonucleoside methylphosphonates, were synthesized. The base sequences of these compounds, d(ApGpGp), d(ApGpGp)2 and d[(ApGpGp)2T], are complementary to the Shine-Dalgarno sequence (-A-C-C-U-C-C-U-) found at the 3'' end of bacterial 16S rRNA. These nonionic oligonucleotide analogs were tested for their ability to inhibit the in vitro translation of mRNA in cell-free systems of E. coli and rabbit reticulocyte. In the E. coli system, d(ApGpGp)2 and d[(ApGpGp)2T] effectively inhibited MS-2 RNA-directed protein synthesis but they had much less effect on 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)2[3H]T] binds to 70S ribosomes (association constant, 2.0 .times. 104 M-1, 37.degree. C) together with the specificity of the inhibitory action of these compounds on protein synthesis strongly suggests that inhibition of translation is a consequence of analog binding to the Shine-Dalgarno sequence of 16S rRNA. The oligonucleoside methylphosphonates inhibited 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. 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 analogs had negligible inhibitory effects on colony formation by transformed human cells. This class of nonionic oligonucleotide analogs may be used to probe and regulate the function and structure of nucleic acids of defined sequence within living cells.

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