Ribozyme vesicles
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.
| 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 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).
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.
http://pubs.acs.org/isubscribe/journals/jacsat/127/i38/figures/ja051784pf00004.gif
