Kathleen R. Blake

Nonionic Oligonucleotide Analogs as New Tools for Studies on the Structure and Function of Nucleic Acids Inside Living Cells

Two types of nonionic oligonucleotide analogs, deoxyribonucleotide alkyl phosphotriesters and deoxyribooligonucleoside methylphosphonates, have been synthesized to serve as selective inhibitors of cellular nucleic acid function. The backbones of these analogs are resistant to nuclease hydrolysis and the analogs are taken up by mammalian cells and certain bacterial cells in culture. Sequence specific analogs inhibit tRNA aminoacylation and translation of mRNA in both mammalian and bacterial cell-free systems in a specific manner as a result of oligomer binding to complementary sequences of the target nucleic acid. These analogs also inhibit cellular protein synthesis and growth of living cells. Selective inhibition of bacterial versus mammalian cell growth is observed with a methylphosphonate oligomer complementary to the Shine-Dalgarno sequence of 16S rRNA. Methylphosphonate complementary to the 5’-end of U1RNA and to the donor splice site of SV40 large T antigen pre-mRNA inhibit T-antigen production in SV40-infected cells.

Control of Gene Expression by Oligonucleoside Methylphosphonates

A major goal in understanding the processes of aging, cancer and differentiation is to understand gene expression and thus the function of various proteins in the overall biochemical processes of the cell. One of the classical ways to study gene expression is through the use of temperature sensitive mutants. Although this approach has been particularly effective in studying gene expression in bacteria and viruses, it is technically more difficult in eukaryotes, particularly mammalian cells. It would be desirable to have an alternative approach which would allow selective inhibition of gene expression either at the level of transcription or at the mRNA level. Recent studies have shown that such regulation may be achieved through the use of complementary DNAs (cDNAs) or anti-sense RNAs. The expression of mRNA can be regulated both in the test tube and in cells by cDNAs which selectively hybridize to a target mRNA. Control of cell-free mRNA translation in this manner is termed hybridization arrest (1). This procedure has been used to study the location and arrangement of adenovirus 2 genes within the viral genome and to analyze mRNA populations in mouse liver (2). Hybridization arrest has also been used to study the function of the 3′-non-coding region of globin mRNA (3).