Efficient stimulation of site-specific ribosome frameshifting by antisense oligonucleotides

Abstract

Evidence is presented that morpholino, 2'-O-methyl, phosphorothioate, and RNA antisense oligonucleotides can direct site-specific -1 translational frameshifting when annealed to mRNA downstream from sequences where the P- and A-site tRNAs are both capable of repairing with -1 frame codons. The efficiency of ribosomes shifting into the new frame can be as high as 40%, determined by the sequence of the frameshift site, as well as the location, sequence composition, and modification of the antisense oligonucleotide. These results demonstrate that a perfect duplex formed by complementary oligonucleotides is sufficient to induce high level -1 frameshifting. The implications for the mechanism of action of natural programmed translational frameshift stimulators are discussed.

FIGURE 1.

Morpholino-induced frameshifting on the UUUUUUA frameshift site. p2LucU₆A was transcribed and translated in rabbit reticulocyte lysate in the absence or presence of increasing amounts of antisense MOAB morpholino. Control (Contr.) morpholino is a morpholino oligonucleotide corresponding to the sense sequence of MOAB. SDS PAGE (4%–12% Bistris polyacrylamide gel) of ³⁵S-methionine-labeled protein products from transcription and translation reactions is shown. Concentrations are shown in micromolar and the location of the full-length frameshift product and nonframeshift termination product are indicated. Average percent frameshifting (% F.S.) and standard deviations (+/−) from the mean are shown below each lane of the gel.

FIGURE 2.

Spacer length effect on morpholino-induced frameshifting. p2LucU₆A was transcribed and translated in rabbit reticulocyte lysate in the absence or presence of increasing amounts of antisense morpholinos MOAD, MOAC, MOAB, MOAA, or MOA-1 that anneal 7, 5, 3, 1, or −1 nt downstream from the frameshift site, respectively. Control (Contr.) morpholino is a morpholino oligonucleotide corresponding to the sense sequence of MOAB. SDS PAGE (4%–12% Bistris polyacrylamide gel) of ³⁵S-methionine-labeled protein products from transcription and translation reactions is shown. Each morpholino was included at a concentration of 1 μM. The location of the full-length frameshift product and nonframeshift termination product are indicated. Average percent frameshifting (% F.S.) and standard deviations (+/−) from the mean are shown below each lane of the gel.

FIGURE 3.

Spacer length effect on morpholino-induced frameshifting. Plasmids p2LucU₆A-0, p2LucU₆A, p2LucU₆A-6, and p2LucU₆A-9 were transcribed and translated in rabbit reticulocyte lysate in the absence or presence of increasing amounts of antisense morpholino MOAB that anneals 0, 3, 6, and 9 nt downstream from the frameshift site for each construct, respectively. SDS PAGE (4%–12% Bistris polyacrylamide gel) of ³⁵S-methionine-labeled protein products from transcription and translation reactions is shown. MOAB was included at a concentration of 1 μM. The location of the full-length frameshift product and nonframeshift termination product are indicated. Average percent frameshifting (% F.S.) and standard deviations (+/−) from the mean are shown below each lane of the gel.

FIGURE 4.

Specificity of morpholino-induced frameshifting. p2LucU₆A was transcribed and translated in rabbit reticulocyte lysate in the absence or presence of antisense morpholinos, MOAB, MOA dmm3, MOA dmm4, or MOA dmm5 that contain 0, 3, 4, or 5 mismatches, respectively. Each morpholino is added at a concentration of 1 μM. Control (Contr.) morpholino is a morpholino oligonucleotide corresponding to the sense sequence of MOAB. SDS PAGE (4%–12% Bistris polyacrylamide gel) of ³⁵S-methionine-labeled protein products from transcription and translation reactions is shown. The location of the full-length frameshift product and nonframeshift termination product are indicated. Average percent frameshifting (% F.S.) and standard deviations (+/−) from the mean are shown below each lane of the gel.

FIGURE 5.

The effect of sequence composition on morpholino induced frameshifting. p2LucU₆A, p2LucU₆A A:T, and p2LucU₆A G:C were transcribed and translated in rabbit reticulocyte lysate in the absence or presence of the complementary antisense morpholinos, MOAB, MOA A:T, and MOA G:C, respectively. Control (Contr.) morpholino is a morpholino oligonucleotide corresponding to the sense sequence of MOAB. Each morpholino is added at a concentration of 1 μM. (IF) p2LucU₆A in which the six Us have been deleted. SDS PAGE (4%–12% Bistris polyacrylamide gel) of ³⁵S-methionine-labeled protein products from transcription and translation reactions is shown. The location of the full-length frameshift product and non-frameshift termination product are indicated. Average percent frame-shifting (% F.S.) and standard deviations (+/−) from the mean are shown below each lane of the gel.

FIGURE 6.

The effect of the heptanucleotide motif on morpholino-induced frameshifting. p2LucAAAUUUA, p2LucGGGAAAC, p2LucUUUAAAC, p2LucAAAAAAC, p2LucAAAAAAG, and p2LucAAAAAAU were transcribed and translated in rabbit reticulocyte lysate in the presence of the complementary antisense morpholino, MOAB. Control © morpholino is a morpholino oligonucleotide corresponding to the sense sequence of MOAB. Each morpholino is added at a concentration of 1 μM. SDS PAGE (4%–12% Bistris polyacrylamide gel) of ³⁵S-methionine-labeled protein products from transcription and translation reactions is shown. The location of the full-length frameshift product and nonframeshift termination product are indicated. Average percent frameshifting (% F.S.) and standard deviations (+/−) from the mean are shown below each lane of the gel.

FIGURE 7.

The effect of antisense oligonucleotide chemistry on frameshift induction. p2lucU₆A was transcribed and translated in rabbit reticulocyte lysate in the presence of increasing amounts of complementary RNA, phosphorothioate, or 2′-O-methyl antisense oligonucleotides. SDS PAGE (4%–12% Bistris polyacrylamide gel) of ³⁵S-methionine-labeled protein products from transcription and translation reactions is shown. The concentration of oligonucleotide (μM) is indicated above each gel lane, and average percent frameshifting (% F.S.) and standard deviations (+/−) from the mean are shown below each lane of the gel.