Antiswitches - Revision history

WikiSysop: /* From Concept to Wet Lab */

2007-12-11T14:51:37Z

‎From Concept to Wet Lab

WikiSysop at 14:41, 11 December 2007

2007-12-11T14:41:47Z

Erzwack: /* From Concept to Wet Lab */

2007-12-06T20:13:36Z

‎From Concept to Wet Lab

Erzwack: /* From Concept to Wet Lab */

2007-12-06T19:54:50Z

‎From Concept to Wet Lab

Erzwack: /* From Concept to Wet Lab */

2007-12-06T19:47:04Z

‎From Concept to Wet Lab

Erzwack: /* From Concept to Wet Lab */

2007-12-06T19:45:06Z

‎From Concept to Wet Lab

Erzwack: /* Design */

2007-12-06T19:44:15Z

‎Design

Erzwack: /* Design */

2007-12-06T19:43:01Z

‎Design

Erzwack: /* References */

2007-12-06T19:41:21Z

‎References

Erzwack at 19:41, 6 December 2007

2007-12-06T19:41:07Z

← Older revision		Revision as of 14:51, 11 December 2007
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−	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).	+	Finally, antiswitches in eukaryotes can be combined to regulate multiple genes for different conditions like the [[Riboregulators\|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).

	[[Image:Combinatorial.JPG]]		[[Image:Combinatorial.JPG]]

← Older revision		Revision as of 14:41, 11 December 2007
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−	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 ~~gene~~ expression in the presence of the ligand while off-switches turn off ~~gene~~ 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, 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.

	==Design==		==Design==

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 ==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 fluoresence 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.
+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).
@@ Line 57: / Line 57: @@
 (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 fluoresence.  The off-switch follows the opposite path as shown in figure 6.
+'''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.

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 (Bayer and Smolke 2005 Permission Pending)
-'''Figure 6:''' The red line represents the tetracycline controlled switch while blue line represents the theophylline controlled switch.  When the concentration of ligand is high enough, enough switches are active and binding to mRNA to stop translation of the gene.
+'''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.
@@ Line 57: / Line 57: @@
 (Bayer and Smolke 2005 Permission Pending)
-'''Figure 7:''' The red line represents the onswitch while the blue Line represents the offswitch.  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.  The off-switch follows the opposite path as in figure 6.
+'''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 fluoresence.  The off-switch follows the opposite path as shown in figure 6.
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 (Bayer and Smolke 2005 Permission Pending)
-'''Figure  8:''' a) On the left, theophylline activates the antiswitch's aptamer, which then binds to the GFP transcript and prevents translation.  On the right, tetracycline binds to the antiswitch's aptamer, 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)is 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.
+'''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.
 ==Modularity==

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 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 fluoresence 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 theophylline and tetracycline binding aptamers worked, the versatility of antiswitches is supported (figure 6).
+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).
 [[Image:Offswitch_experiment.JPG]]

← Older revision		Revision as of 19:44, 6 December 2007
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−	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 ~~bids~~ to the aptamer; thus, the antisense stem can duplex and is no longer free to bind to the transcript.	+	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.

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-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.
+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).
 [[Image:Caffeine.JPG]]
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-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.
+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.  In an on-switch, the aptamer swings away from the antisense stem when the ligand bids to the aptamer; thus, the antisense stem can duplex and is no longer free to bind to the transcript.
+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 bids to the aptamer; thus, the antisense stem can duplex and is no longer free to bind to the transcript.

← Older revision		Revision as of 19:41, 6 December 2007
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	Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.		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. [http://www.pnas.org/cgi/content/abstract/104/36/14283 Abstract].
		+

	[[Post-transcriptional Regulation Technologies - Erin Zwack\|Return to Post-transcriptional Regulation Technologies]]		[[Post-transcriptional Regulation Technologies - Erin Zwack\|Return to Post-transcriptional Regulation Technologies]]

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-Antiswitches, trans-RNA molecules that regulate translation of mRNA based on 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 gene expression in the presence of the ligand while off-switches turn off gene expression in the presence of 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, 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 gene expression in the presence of the ligand while off-switches turn off gene 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.
 ==Design==
@@ Line 12: / Line 12: @@
 (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 depending on whether the aptamer is bound to its ligand.
+'''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.
@@ Line 23: / Line 23: @@
-While Smolke and Bayer mainly used the aptamer for the ligand theophylline, aptamers for many other molecules are now being generated using rational design.  The large number of possible aptamers provides versatility in what will control gene expression.
+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.