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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 approximately 11 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 Phosphorodiamidate Morpholino Oligomer Length and Target Position Effects on Gene-Specific Inhibition inEscherichia coli