Ribozyme vesicles - Revision history

WikiSysop: /* Experimental Design */

2007-12-11T16:25:54Z

‎Experimental Design

WikiSysop: /* Experimental Design */

2007-12-11T16:23:22Z

‎Experimental Design

WikiSysop: /* Goals */

2007-12-11T16:20:34Z

‎Goals

Dajordan: /* Results */

2007-12-08T19:18:57Z

‎Results

Dajordan: /* Experimental Design */

2007-12-08T19:17:59Z

‎Experimental Design

Dajordan: /* Goals */

2007-12-06T21:38:33Z

‎Goals

Dajordan at 21:38, 6 December 2007

2007-12-06T21:38:21Z

Dajordan at 21:09, 6 December 2007

2007-12-06T21:09:03Z

Dajordan: /* Experimental Design */

2007-12-06T21:07:00Z

‎Experimental Design

Dajordan at 21:05, 6 December 2007

2007-12-06T21:05:27Z

@@ Line 21: / Line 21: @@
 ''Table 1 was re-created using data from Chen ''et al.'', 2005.''
-The stability in the presence of Mg<sup>2+</sup> 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 Mg<sup>2+</sup> on the permeability of the vesicles. First, they needed to address whether Mg<sup>2+</sup> 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 Mg<sup>2+</sup> 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 Mg<sup>2+</sup> 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.
+The stability in the presence of Mg<sup>2+</sup> 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 Mg<sup>2+</sup> on the permeability of the vesicles. First, they needed to address whether Mg<sup>2+</sup> 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 Mg<sup>2+</sup> 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 Mg<sup>2+</sup> 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 out 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 Granted by Jack Szostak.''
+Figure 1. (A) Leakage of encapsulated calcein, a fluorescent dye, was measured over time with or without 4 mM MgCl<sub>2</sub>, 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 Mg<sup>2+</sup> versus the control without Mg<sup>2+</sup> (black line). (D) Leakage of encapsulated H-UMP vesicles was measured over time in response to MgCl<sub>2</sub> (red) versus the control (black) without MgCl<sub>2</sub>. ''Image Permission Granted by Jack Szostak.''
 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.

@@ Line 18: / Line 18: @@
 |}
-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. To test the stability of various composititons of MA and GGM, investigators monitored dye retention in the vesicle <1 h after addition of MgCl<sub>2</sub>. The concentration of MgCl<sub>2</sub> that caused leakage to occur is defined as the maximum concentrated tolerated by the vesicle. An additional measure of the maximum concentration of MgCl<sub>2</sub> 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.''
+''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.
+The stability in the presence of Mg<sup>2+</sup> 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 Mg<sup>2+</sup> on the permeability of the vesicles. First, they needed to address whether Mg<sup>2+</sup> 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 Mg<sup>2+</sup> 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 Mg<sup>2+</sup> 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

@@ Line 1: / Line 1: @@
 == 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).
+*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.

@@ Line 42: / Line 42: @@
 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.''
+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 Granted by Jack Szostak.''
 == Conclusions and Further Experiments ==

@@ Line 25: / Line 25: @@
 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.''
+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 Granted by Jack Szostak.''
 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.

← Older revision		Revision as of 21:09, 6 December 2007
Line 30:		Line 30:

	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.		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 ==		== Results ==

@@ Line 29: / Line 29: @@
 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 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.
+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.
 == Results ==

← Older revision		Revision as of 21:05, 6 December 2007
Line 28:		Line 28:

	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.		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 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.

	== Results ==		== Results ==