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All information about antiswitches came from Bayer and Smolke (2005).

Antiswitches, trans-RNA molecules that regulate translation of mRNA based on the presence or absence of specific ligands, were first developed by Smolke and Bayer (2005) in Saccharomyces cerevisiae. Two types of antiswitches were engineered: on-switches and off-switches. On-switches turn on protein expression in the presence of the ligand while off-switches turn off protein expression in the presence of the ligand. Control by the ligand allows researchers to regulate pathways’ protein production with less leakiness, low level transcription even when “off”, than using a specific promoter.


Antiswitches are made of an aptamer and two stems: the aptamer stem and the antisense stem (figure 3).


(Bayer and Smolke 2005 Permission Pending)

Figure 3: The antisense stem can bind to itself (duplex). The antisense stem also contains the complement to the RNA that a researcher desires to regulate. The aptamer stem swings either toward the antisense stem and disrupts the duplex or swings away from the antisense stem and allows the antisense stem to duplex with itself depending on whether the aptamer is bound to its ligand.

The aptamer is the sequence that binds the ligand and causes a conformational (shape) change of the antiswitch molecule. Aptamers can be highly specific for their particular molecules. The theophylline aptamer used by Smolke and Bayer can distinguish between caffeine and theophylline, which differ by a single methyl (figure 4).


Figure 4. Caffeine (a) and Theophylline (b) differ by one methyl group (circled in red).

While Smolke and Bayer mainly used the aptamer for the ligand theophylline, aptamers for many other molecules are now being generated using rational design (Win and Smolke 2007). The large number of possible aptamers provides versatility in what will control gene expression.

The antisense stem contains a sequence that complements a targeted RNA transcript and a second sequence that sequesters this complementary sequence to keep it from binding the transcript. When the antisense stem is not duplexed with itself, it prevents translation by binding to the complementary mRNA (figure 5).

The aptamer stem is a short sequence that complements a portion of the sequestering sequence of the antisense stem. In an off-switch, the aptamer stem swings towards the antisense stem and displaces the portion that complements the targeted transcript when the liand binds to the aptamer (figure 5). In an on-switch, the aptamer swings away from the antisense stem when the ligand binds to the aptamer; thus, the antisense stem can duplex and is no longer free to bind to the transcript.


(Bayer and Smolke 2005 Permission Pending)

Figure 5: a) An inactive off-switch. The antisense stem is duplexed with itself. b) The same switch after being activated by theophylline (blue ellipse). The antisense stem is duplexed with the mRNA; thus, translation is stopped.

From Concept to Wet Lab

Smolke and Bayer synthesized genes for antiswitches with antisense stems that contained complements to either GFP or YFP transcripts and the aptamer to either theophylline or tetracycline. These genes were then transformed into Saccharomyces cerevisiae. Several experiments, including dose-response curves to the appropriate ligand and fluorescence measurements of cells when exposed to both the appropriate ligand and wrong ligand, demonstrated that these antiswitches effectively regulate translation of their specific target in response to only the correct ligand.

In the dose-response experiments for off-switches when enough ligand (~ 1 mM theophylline or tetracycline depending on the switch) was present, the inactive off-switches became active and relative GFP expression dropped to almost zero. These experimental results support the antiswitch technology being functional. As off-switches with either theophylline or tetracycline aptamers worked, the versatility of antiswitches is supported (figure 6).

Offswitch experiment.JPG

(Bayer and Smolke 2005 Permission Pending)

Figure 6: The red line represents the tetracycline controlled off-switch while blue line represents the theophylline controlled off-switch. When the concentration of ligand is high enough, enough off-switches are active and binding to mRNA to stop translation of the gene.

In the dose-response experiments for the on-switches when ~ 1 mM of theophylline was added to cells containing the inactive on-switch, the cells relative expression of GFP jumped from near zero to approximately 0.9. This data show that the on-switch is also functional (figure 7).

Onswitch experiment.JPG

(Bayer and Smolke 2005 Permission Pending)

Figure 7: The red line represents the on-switch while the blue Line represents the off-switch. When a high enough concentration of ligand is present, enough on-switches are activated and have thus released their target mRNA for detectable near normal translation to occur as measured by fluorescence. The off-switch follows the opposite path as shown in figure 6.

Finally, antiswitches in eukaryotes can be combined to regulate multiple genes for different conditions like the taRNA/crRNA system in prokaryotes. By simply switching the aptamer and antisense stem targeting sequence, the switch now controls a different gene by a different stimulus. Off-switch experiments using a theophylline aptamer with a GFP targeting antisense stem and a tetracycline aptamer with a YFP targeting antisense stem showed that the switches regulate the genes independently and can be used in combination to create complex regulatory mechanisms (figure 8).


(Bayer and Smolke 2005 Permission Pending)

Figure 8: a) On the left, theophylline binds to the antiswitch's aptamer and activates the off-switch, which then binds to the GFP transcript and prevents translation. On the right, tetracycline binds to the antiswitch's aptamer and activates the off-switch, which then binds to the YFP transcript and prevents translation. If only one ligand is present (either theophylline or tetracycline), only one gene's transcripts (either GFP or YFP) are not translated. b) When no theophylline or tetracycline is present, both GFP and YFP are expressed at near normal levels. When only tetracycline is present, GFP is expressed at near normal levels while YFP expression is close to zero. When only theophylline is present, YFP is expressed at near normal levels while GFP expression is close to zero. When both tetracycline and theophylline are present, GFP and YFP expression are both close to zero.


While antiswitches have many advantages, they do lack modularity, the ability to be integrated with any gene without redesigning a new sequence specific part (Isaacs et al. 2004). If you want to regulate a new gene, you have to synthesize a whole new antiswitch because the antisense stem is specific to a particular gene and the aptamer stem is specific to a particular antisense stem.


Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat Biotechnol. (2005) 3:337-43.

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

Win MN, Smolke CD (2007). A modular and extensible RNA-based gene-regulatory platform for engineering cellular function. PNAS 104(36):14283-8. Epub 2007 Aug 20. Abstract.

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