== Goals ==
*Create the "simplest possible protocell" capable of having a self-replicating informational molecule and a mechanism for spatial localization such as compartmentalization (Chen et al. 2005).
*Use membrane boundary that can grow and divide with being too complex and that can allow passive diffusion of ion and substrates
*Encapsulation of catalytic (self-replicating) RNA molecules within self-replicating membrane vesicles.

== Experimental Design ==
A unique and beneficial aspect of fatty acid vesicles is that they have autocatalytic growth and can repeatedly divide on their own. The first issue addressed is to create membranes that are stable but can allow passive diffusion of ions and substrates in and out of the vesicle. The reason that this aspect of the protocell is so essential is because the formation of RNA catalysts requires the addition of magnesium ions to create the tertiary structure of the ribozyme. To accomplish this goal, researchers observed the effects of magnesium on the stability and permeability of vesicles consisting of fatty acids known as myristoleic acid (MA) and glycerol monomyristoleate (GMM). Thus, they experimented with different ratios of MA to GMM to increase tolerance of Mg2+ in vesicles and allow for passive diffusion.

{| border="1"
|+ Table 1. MgCl2 Tolerance of Simple Vesicles
! MA:GMM ratio !! [MgCl2] tolerated, assayed by dye leakage (mM) !! [MgCl2] at turbidity change (mM)
|-
| 1:0 || 0.5 || 1
|-
| 4:1 || 2 || 3
|-
| 2:1 || 4 || 6
|}

Table 1. To test the stability of various composititons of MA and GGM, investigators monitored dye retention in the vesicle <1 h after addition of MgCl2. The concentration of MgCl2 that caused leakage to occur is defined as the maximum concentrated tolerated by the vesicle. An additional measure of the maximum concentration of MgCl2 allowed by the vesicle is using the [http://en.wikipedia.org/wiki/Turbidity turbidity] to access the cloudiness created by individual particles.
''Table 1 was re-created using data from Chen et al. 2005.''

The stability in the presence of Mg2+ was shown to increase as the proportion of GMM increased. However, higher proportions than 2:1 MA to GMM resulted in "the appearance of oil droplets mixed with vesicles" (Chen et al. 2005). Then, researchers were interested in testing the effects of Mg2+ on the permeability of the vesicles. First, they needed to address whether Mg2+ caused permanent permeability in vesicles. Therefore, they measured the percent of dye leakage of vesicles over time. Dye leakage was found to increase over time in a period of one day, showing that permeability of the vesicle exists permanently throughout the experiment (Figure 1A and 1B). Then, researchers tested whether "large-scale destabilization" occurs in vesicles due to Mg2+ by measuring presence of RNA decamers tagged with fluroescent labels (Chen et al. 2005). They would expect if destabilization occurs then the RNA would leak out of the vesicles but instead they found that RNA remained in the vesicles (Figure 1C). However, a mononucleotide (H-UMP) of RNA was found to be permeable in the same conditions (Figure 1D). The paper attributes this difference between the permeability of mononucleotide of RNA and larger RNA molecule to neutralization of negative charges in the RNA and stabilization caused by Mg2+ of the membrane and solute interactions, which would prevent RNA molecules from leaking. Another reason not mentioned in the paper could be that larger RNA molecules may be too large to efficiently diffuse of the vesicles whereas smaller RNA mononucleotides may be able to pass through the semi-permeable membrane.

http://pubs.acs.org/isubscribe/journals/jacsat/127/i38/figures/ja051784pf00001.gif

Figure 1. (A) Leakage of encapsulated calcein, a fluorescent dye, was measured over time with or without 4 mM MgCl2, represented by the blue and black lines, respectively. (B) Fractions of encapsulated versus free calcein that has leaked out of the vesicle at 22 hr. (C) Leakage of encapsulated RNA decamer is shown by the difference between encapsulated and free RNA using size-exclusion chromatography after 19 hr. The red line represents response to 4 mM Mg2+ versus the control without Mg2+ (black line). (D) Leakage of encapsulated H-UMP vesicles was measured over time in response to MgCl2 (red) versus the control (black) without MgCl2. ''Image Permission Pending.''

In addition, investigators used similar processes by using a fluorescent dye sensitive to magnesium known as magfura-2 to verify that these vesicles were indeed permeable to magnesium.

Lastly, researchers attempted to increase vesicle growth by addition of [http://en.wikipedia.org/wiki/Micelle micelles] to vesicles. It resulted in a ~50% growth in the surface area of the vesicle. Additionally, dodecane is added as a hydrophobic spacer, resulting in 2:1:0: MA:GMM:dodecane micelles. Thus the overall growth of these micelles to vesicles of the same composition was 40% in one equivalent of micelle.

http://upload.wikimedia.org/wikipedia/commons/thumb/c/c6/Phospholipids_aqueous_solution_structures.svg/250px-Phospholipids_aqueous_solution_structures.svg.png

[http://upload.wikimedia.org/wikipedia/commons/thumb/c/c6/Phospholipids_aqueous_solution_structures.svg/250px-Phospholipids_aqueous_solution_structures.svg.png Image Source]

== Results ==
'''"Ribozyme Activity in Simple Vesicles"(Chen et al., 2005)'''

Vesicles of 2:1:0.3 MA:GMM:dodecane were created to encapulate self-cleaving hammerhead ribozymes. This ribozyme (N15min7) is important because it can both cleave and ligate RNA, which will be very important for simple cell-like structures. When Mg2+ is added, the ribozyme cleaves itself into two smaller fragments. The fraction of ribozymes cleaved over time when exposed to 4 mM MgCl2 increased to about 0.66 in unencapsulated vesicles (Figure 2A) and 0.60 in encapsulated vesicles (Figure 2B). The top band on the gel represent the uncleaved ribozymes, while the bottom band represents the cleaved ribozyme, and the lanes correspond with each time point. As the fraction of uncleaved ribozymes decreases, the fraction of cleaved ribozymes increases, which is what we would expect. The vesicles were very stable because even after 15 minutes of exposure to MgCl2, the vesicles remained encapsulated (Figure 2C).

http://pubs.acs.org/isubscribe/journals/jacsat/127/i38/figures/ja051784pf00004.gif

Figure 2. (A and B) The self-cleavage activity of ribozyme N15min7 measured by the fraction cleaved over time. The insets on the graph are phoshorimages of the assay gels. (A) represents unencapsulated ribozymes while (B) represents encapsulated MA:GMM:dodecane ribozymes. (C) Size-exclusion chromatography of MA:GMM:dodecane vesicles of "radiolabeled N15min7 RNA remained encapsulated 15 min after ther addition of MgCl2" (Chen et al., 2005). ''Image Permission Pending.''

== Conclusions and Further Experiments ==
Therefore, these researchers sucessfully created vesicles that are permeable to ions and substrates necessary for proper ribozyme function and showed that catalytic ribozyme activity can occur inside these vesicles without any significant loss of functionality. These novel cell-like vesicles open the doors to exploring new ways of engineering and understanding biological systems.

<hr>

== '''[http://gcat.davidson.edu/GcatWiki/index.php/Applications_of_Ribozymes_in_Synthetic_Systems_-_Danielle_Jordan Main Page]''' ==

Ribozyme Switch

2007-12-06T21:37:18Z

Dajordan:

Strand-Displacement

2007-12-06T21:36:44Z

Dajordan:

The strand-displacement mechanism uses competitive binding of two identical nucleic acid sequences, the competing strand and the switching strand. It is based on rational design and results in the disruption or restoration of the hammerhead ribozyme as a result of restoration in the aptamer domain [http://www.pnas.org/cgi/content/full/0703961104/DC1#ST (Supplementary Information)].
== Competing strand ==
The competing strand is the nucleic acid sequence that is bound to the the general transmission region in the restored switch conformation in the presence of a ligand [http://www.pnas.org/cgi/content/full/0703961104/DC1#ST (Supplementary Information)].

== Switching strand ==
The switching strand is the nucleic acid sequence that is bound to the general transmission region in the disrupted switch conformation in the absense of a ligand [http://www.pnas.org/cgi/content/full/0703961104/DC1#ST (Supplementary Information)].

== ON and OFF Ribozyme Switches ==
Win and Smolke designed ON and OFF switches by the strand-displacement mechanism that allows either disruption or activation of the ribozyme catalytic core. The ON switch in Figure 2A begins with a ribozyme L2bulge1 that starts out in the active conformation with the aptamer unbound. When the aptamer is unbound, the catalytic core is not disrupted, which allows the ribozyme to self-cleave. The cleaving effect of the ribozyme causes down regulation of gene expression. By a simple nucleotide shift when the competing strand binds, the conformation of the aptamer where the ligand can bind changes, allowing theophilline (the ligand) to bind. When theophilline binds, the conformation of the catalytic core has a bulge in it, which prevents self-cleavage. Thus, the gene can now be up regulated. Ideally the two conformations of bound and unbound states would be constantly changing from one to the other, but when theophilline binds, it stays bound, which shifts the equilibrium so that more ribozymes end up in the ON state (Figure 2A). The researchers also show how the amount of gene expression is dependent on theophilline concentration and follows a dose response pattern (Figure 2C).
 
In much the same way, the OFF switch begins with a ribozyme that has a bulge and therefore is inactive and allows gene expression. When nucleotide shifting occurs, the aptamer is bound with theophilline and the ribozyme is allowed to cleave itself, resulting in the OFF state. Once again, the OFF switch has a dose response curve due to theophilline concentrations, which is important in determining gene regulation effects (Figure 1D).
http://www.pnas.org/content/vol104/issue36/images/large/zpq0340773700002.jpeg

Figure 2. "Regulatory properties of the strand-displacement information transmission mechanism. The color scheme corresponds to that used in Fig. 1 with the following exceptions: switching strand, red; competing strand, green. (A) Gene expression ON ribozyme switch platform, L2bulge1. (B) Gene expression OFF ribozyme switch platform, L2bulgeOff1. (C and D) The theophylline-dependent gene-regulatory behavior of L2bulge1 (ON switch) (C), L2bulgeOff1 (OFF switch) (D), and L2Theo (nonswitch control). Gene-expression levels are reported in fold as defined in SI Text and were normalized to the expression levels in the absence of effector" (Win and Smolke, 2007). ''Image Permission Pending.''

== Tunability of Ribozyme Switches ==
Next, the investigators wanted to show that the ON and OFF switches created could be practical and applicable because they can be rationally designed to exhibit different levels of gene regulation. The swithes were created the same way as before except that they made many different aptamers that can be used to elicit differents responses of induction in fold of GFP due at 5 mM of theophylline (Figure 4B). The variations in inductions can be explained by the differences in energetics between the two states so that the aptamers with the least energy difference between the bound and unbound state have the highest induction [http://www.pnas.org/cgi/content/full/0703961104/DC1#T2 Supplementary Information].
http://www.pnas.org/content/vol104/issue36/images/large/zpq0340773700004.jpeg

Figure 3. "Tunability of the strand-displacement-based ribozyme switches. (A) Sequences targeted by the rational tuning strategies are indicated in the dashed boxes on the effector-bound conformations of L2bulge1 (ribozyme-inactive) and L2bulgeOff1 (ribozyme-active). (B and C) Regulatory activities of tuned strand-displacement-based ON (B) and OFF (C) ribozyme switches. Gene-regulatory effects of these switches at 5 mM theophylline are reported in fold induction for ON switches and fold repression for OFF switches relative to the expression levels in the absence of theophylline as described in Fig. 2" (Win and Smolke, 2007). ''Image Permission Pending.''

== Links ==
[[Ribozyme Switch]]

== '''[http://gcat.davidson.edu/GcatWiki/index.php/Applications_of_Ribozymes_in_Synthetic_Systems_-_Danielle_Jordan Main Page]''' ==

2007-12-06T20:37:12Z

Dajordan:

== Goals ==
*Create the "simplest possible protocell" capable of having a self-replicating informational molecule and a mechanism for spatial localization such as compartmentalization (Chen et al. 2005).
*Use membrane boundary that can grow and divide with being too complex and that can allow passive diffusion of ion and substrates
*Encapsulation of catalytic (self-replicating) RNA molecules within self-replicating membrane vesicles.

== Experimental Design ==
A unique and beneficial aspect of fatty acid vesicles is that they have autocatalytic growth and can repeatedly divide on their own. The first issue addressed is to create membranes that are stable but can allow passive diffusion of ions and substrates in and out of the vesicle. The reason that this aspect of the protocell is so essential is because the formation of RNA catalysts requires the addition of magnesium ions to create the tertiary structure of the ribozyme. To accomplish this goal, researchers observed the effects of magnesium on the stability and permeability of vesicles consisting of fatty acids known as myristoleic acid (MA) and glycerol monomyristoleate (GMM). Thus, they experimented with different ratios of MA to GMM to increase tolerance of Mg2+ in vesicles and allow for passive diffusion.

{| border="1"
|+ Table 1. MgCl2 Tolerance of Simple Vesicles
! MA:GMM ratio !! [MgCl2] tolerated, assayed by dye leakage (mM) !! [MgCl2] at turbidity change (mM)
|-
| 1:0 || 0.5 || 1
|-
| 4:1 || 2 || 3
|-
| 2:1 || 4 || 6
|}

Table 1. To test the stability of various composititons of MA and GGM, investigators monitored dye retention in the vesicle <1 h after addition of MgCl2. The concentration of MgCl2 that caused leakage to occur is defined as the maximum concentrated tolerated by the vesicle. An additional measure of the maximum concentration of MgCl2 allowed by the vesicle is using the [http://en.wikipedia.org/wiki/Turbidity turbidity] to access the cloudiness created by individual particles.
''Table 1 was re-created using data from Chen et al 2005.''

The stability in the presence of Mg2+ was shown to increase as the proportion of GMM increased. However, higher proportions than 2:1 MA to GMM resulted in "the appearance of oil droplets mixed with vesicles" (Chen et al. 2005). Then, researchers were interested in testing the effects of Mg2+ on the permeability of the vesicles. First, they needed to address whether Mg2+ caused permanent permeability in vesicles. Therefore, they measured the percent of dye leakage of vesicles over time. Dye leakage was found to increase over time in a period of one day, showing that permeability of the vesicle exists permanently throughout the experiment (Figure 1A and 1B). Then, researchers tested whether "large-scale destabilization" occurs in vesicles due to Mg2+ by measuring presence of RNA decamers tagged with fluroescent labels (Chen et al. 2005). They would expect if destabilization occurs then the RNA would leak out of the vesicles but instead they found that RNA remained in the vesicles (Figure 1C). However, a mononucleotide (H-UMP) of RNA was found to be permeable in the same conditions (Figure 1D). The paper attributes this difference between the permeability of mononucleotide of RNA and larger RNA molecule to neutralization of negative charges in the RNA and stabilization caused by Mg2+ of the membrane and solute interactions, which would prevent RNA molecules from leaking. Another reason not mentioned in the paper could be that larger RNA molecules may be too large to efficiently diffuse of the vesicles whereas smaller RNA mononucleotides may be able to pass through the semi-permeable membrane.

http://pubs.acs.org/isubscribe/journals/jacsat/127/i38/figures/ja051784pf00001.gif

Figure 1. (A) Leakage of encapsulated calcein, a fluorescent dye, was measured over time with or without 4 mM MgCl2, represented by the blue and black lines, respectively. (B) Fractions of encapsulated versus free calcein that has leaked out of the vesicle at 22 hr. (C) Leakage of encapsulated RNA decamer is shown by the difference between encapsulated and free RNA using size-exclusion chromatography after 19 hr. The red line represents response to 4 mM Mg2+ versus the control without Mg2+ (black line). (D) Leakage of encapsulated H-UMP vesicles was measured over time in response to MgCl2 (red) versus the control (black) without MgCl2.

http://pubs.acs.org/isubscribe/journals/jacsat/127/i38/figures/ja051784pf00004.gif

== Results ==
'''"Ribozyme Activity in Simple Vesicles"'''

Vesicles of 2:1:0.3 MA:GMM:dodecane were created to encapulate self-cleaving hammerhead ribozymes. This ribozyme (N15min7) is important because it can both cleave and ligate RNA, which will be very important for simple cell-like structures. When Mg2+ is added, the ribozyme cleaves itself into two smaller fragments. The fraction of ribozymes cleaved over time when exposed to 4 mM MgCl2 increased to about 0.66 in unencapsulated vesicles (Figure 2A) and 0.60 in encapsulated vesicles (Figure 2B). The vesicles were very stable because even after 15 minutes of exposure to MgCl2, the vesicles remained encapsulated (Figure 2C).

http://pubs.acs.org/isubscribe/journals/jacsat/127/i38/figures/ja051784pf00004.gif

== Conclusions and Further Experiments ==
Therefore, these researchers sucessfully created vesicles that are permeable to ions and substrates necessary for proper ribozyme function and showed that catalytic ribozyme activity can occur inside these vesicles without any significant loss of functionality. These novel cell-like vesicles open the doors to exploring new ways of engineering and understanding biological systems.

<hr>

== '''[http://gcat.davidson.edu/GcatWiki/index.php/Applications_of_Ribozymes_in_Synthetic_Systems_-_Danielle_Jordan Main Page]''' ==

2007-12-06T19:29:49Z

Dajordan: /* Production of Monomeric Ribozymes */

Nanocircles

2007-12-06T19:28:25Z

Dajordan: /* Results */

Strand-Displacement

2007-12-06T19:15:55Z

Dajordan:

The strand-displacement mechanism uses competitive binding of two identical nucleic acid sequences, the competing strand and the switching strand. It is based on rational design and results in the disruption or restoration of the hammerhead ribozyme as a result of restoration in the aptamer domain [http://www.pnas.org/cgi/content/full/0703961104/DC1#ST (Supplementary Information)].
== Competing strand ==
The competing strand is the nucleic acid sequence that is bound to the the general transmission region in the restored switch conformation in the presence of a ligand [http://www.pnas.org/cgi/content/full/0703961104/DC1#ST (Supplementary Information)].

== Switching strand ==
The switching strand is the nucleic acid sequence that is bound to the general transmission region in the disrupted switch conformation in the absense of a ligand [http://www.pnas.org/cgi/content/full/0703961104/DC1#ST (Supplementary Information)].

== ON and OFF Ribozyme Switches ==
Win and Smolke designed ON and OFF switches by the strand-displacement mechanism that allows either disruption or activation of the ribozyme catalytic core. The ON switch in Figure 2A begins with a ribozyme L2bulge1 that starts out in the active conformation with the aptamer unbound. When the aptamer is unbound, the catalytic core is not disrupted, which allows the ribozyme to self-cleave. The cleaving effect of the ribozyme causes down regulation of gene expression. By a simple nucleotide shift when the competing strand binds, the conformation of the aptamer where the ligand can bind changes, allowing theophilline (the ligand) to bind. When theophilline binds, the conformation of the catalytic core has a bulge in it, which prevents self-cleavage. Thus, the gene can now be up regulated. Ideally the two conformations of bound and unbound states would be constantly changing from one to the other, but when theophilline binds, it stays bound, which shifts the equilibrium so that more ribozymes end up in the ON state (Figure 2A). The researchers also show how the amount of gene expression is dependent on theophilline concentration and follows a dose response pattern (Figure 2C).
 
In much the same way, the OFF switch begins with a ribozyme that has a bulge and therefore is inactive and allows gene expression. When nucleotide shifting occurs, the aptamer is bound with theophilline and the ribozyme is allowed to cleave itself, resulting in the OFF state. Once again, the OFF switch has a dose response curve due to theophilline concentrations, which is important in determining gene regulation effects (Figure 1D).
http://www.pnas.org/content/vol104/issue36/images/large/zpq0340773700002.jpeg

Figure 2. "Regulatory properties of the strand-displacement information transmission mechanism. The color scheme corresponds to that used in Fig. 1 with the following exceptions: switching strand, red; competing strand, green. (A) Gene expression ON ribozyme switch platform, L2bulge1. (B) Gene expression OFF ribozyme switch platform, L2bulgeOff1. (C and D) The theophylline-dependent gene-regulatory behavior of L2bulge1 (ON switch) (C), L2bulgeOff1 (OFF switch) (D), and L2Theo (nonswitch control). Gene-expression levels are reported in fold as defined in SI Text and were normalized to the expression levels in the absence of effector" (Win and Smolke 2007).

== Tunability of Ribozyme Switches ==
Next, the investigators wanted to show that the ON and OFF switches created could be practical and applicable because they can be rationally designed to exhibit different levels of gene regulation. The swithes were created the same way as before except that they made many different aptamers that can be used to elicit differents responses of induction in fold of GFP due at 5 mM of theophylline (Figure 4B). The variations in inductions can be explained by the differences in energetics between the two states so that the aptamers with the least energy difference between the bound and unbound state have the highest induction [http://www.pnas.org/cgi/content/full/0703961104/DC1#T2 Supplementary Information].
http://www.pnas.org/content/vol104/issue36/images/large/zpq0340773700004.jpeg

Figure 3. "Tunability of the strand-displacement-based ribozyme switches. (A) Sequences targeted by the rational tuning strategies are indicated in the dashed boxes on the effector-bound conformations of L2bulge1 (ribozyme-inactive) and L2bulgeOff1 (ribozyme-active). (B and C) Regulatory activities of tuned strand-displacement-based ON (B) and OFF (C) ribozyme switches. Gene-regulatory effects of these switches at 5 mM theophylline are reported in fold induction for ON switches and fold repression for OFF switches relative to the expression levels in the absence of theophylline as described in Fig. 2" (Win and Smolke 2007).

== Links ==
[[Ribozyme Switch]]

== '''[http://gcat.davidson.edu/GcatWiki/index.php/Applications_of_Ribozymes_in_Synthetic_Systems_-_Danielle_Jordan Main Page]''' ==