Antisense Phosphorodiamidate Morpholino Oligomer Length and Target Position Effects on Gene-Specific Inhibition inEscherichia coli

Abstract

Phosphorodiamidate morpholino oligomers (PMOs) are synthetic DNA analogs that inhibit gene expression in a sequence-dependent manner. PMOs of various lengths (7 to 20 bases) were tested for inhibition of luciferase expression in Escherichia coli. Shorter PMOs generally inhibited luciferase greater than longer PMOs. Conversely, in bacterial cell-free protein synthesis reactions, longer PMOs inhibited equally or more than shorter PMOs. Overlapping, isometric (10-base) PMOs complementary to the region around the start codon of luciferase inhibited to different extents in bacterial cell-free protein expression reactions. Including the anti-start codon in PMOs was not required for maximal inhibition. PMOs targeted to 5 nontranslated or 3 coding regions within luciferase mRNA did not inhibit, except for one PMO targeted to the ribosome-binding site. Inhibition of luciferase expression correlated negatively with the predicted secondary structure of mRNA regions targeted by PMO but did not correlate with C؉G content of targeted regions. The effects of PMO length and position were corroborated by using PMOs (6 to 20 bases) targeted to acpP, a gene required for viability. Because inhibition by PMOs of ϳ11 bases was unexpected based on previous results in eukaryotes, we tested an 11-base PMO in HeLa cells and reticulocyte cell-free protein synthesis reactions. The 11-base PMO significantly inhibited luciferase expression in HeLa cells, although less than did a 20-base PMO. In reticulocyte cell-free reactions, there was a trend toward more inhibition with longer PMOs. These studies indicate that strategies for designing PMOs are substantially different for prokaryotic than eukaryotic targets.Antisense drugs are short (10 to 25 bases) oligomers that mimic DNA or RNA and inhibit gene expression in a sequence-dependent manner (2, 6, 29, 30). They differ from native RNA or DNA in the chemical structure that links the four common bases.Most of the reported work on antisense drugs has been accomplished in eukaryotic systems. In eukaryotes, antisense compounds inhibit by two general mechanisms. Compounds such as phosphorothioates hybridize to mRNA and promote its degradation by RNase H (3, 5, 28). Other compounds, such as peptide nucleic acid (PNA) and phosphorodiamidate morpholino oligomer (PMO), hybridize to specific mRNA and block translation by an RNase H-independent mechanism (3, 28).The most effective region for targeting PNA and PMO is the 5Ј untranslated region and initiation codon of mRNA (3,10,14,15,18,20,28,30,32). The optimal length of a PNA or PMO depends on achieving a balance between specificity and efficacy and is estimated to be about 15 to 25 bases (2, 14, 16, 21, 30). However, experimental evidence suggests that many factors, such as cellular uptake or invasion of mRNA secondary structure, influence efficacy and can tip the balance in favor of shorter antisense oligomers (13,15,19,23,31).Sequence-specific antibacterial drugs are a recent application of antisense technology (11,12,17,24,25,33). Nielsen and cowork...

Inhibition of Intracellular Growth of Salmonella enterica Serovar Typhimurium in Tissue Culture by Antisense Peptide-Phosphorodiamidate Morpholino Oligomer

“…2B). A similar effect has been observed in cultures of E. coli treated with AcpP PMOs (9,14). There was no significant difference (P Ͼ 0.05) in viable cells of cultures treated with either one of the Scr PPMOs compared to the untreated culture.…”

“…2A), although it significantly reduced the number of viable cells from the 24-h culture. Previous results with AcpP PMOs and cultures of E. coli have also shown a similar effect (9,14). The cause of this effect is unknown.…”

Variations in Amino Acid Composition of Antisense Peptide-Phosphorodiamidate Morpholino Oligomer Affect Potency against Escherichia coli In Vitro and In Vivo

“…The rationale for the length and position of the PMO on the target mRNA has been characterized previously (5). This PMO sequence has been shown to inhibit the growth of E. coli in pure cultures (5,8,23), infected tissue culture (23), and mouse models of infection (8,22). The base sequence of the nonsensical control PMOs is 5Ј-TCTCAGATGGT-3Ј.…”

“…The base sequence of the nonsensical control PMOs is 5Ј-TCTCAGATGGT-3Ј. The nonsensical (scrambled) sequence is a random sequence of the same 11 bases used in the AcpP PMO and has previously been shown not to inhibit the growth of E. coli (5,8,23,24).…”

“…When antisense oligomers are added to pure bacterial cultures, they have been shown to decrease the levels of expression of reporter genes such as luciferase (5,7), activate endogenous genes such as ␤-galactosidase (7), or inhibit growth by targeting essential genes or RNA (9,10,11,14,23,28). In addition, antisense oligomers reduce infection and increase the survival of mice infected with Escherichia coli (8,21,22).…”