Engineering a replicating system in a microscopic bioreactor
Wenonah Vercoutere*, Pierre-Alain Monnard, and David Deamer
Department of Chemistry & Biochemistry, University of California, Santa Cruz
This is an abstract
for a presentation given at the
Foresight Conference on Molecular Nanotechnology.
There will be a link from here to the full article when it is
available on the web.
The origins of self-assembled replicating systems date back over 3.8 billion years. A likely primitive organization would have included a recurring reaction separated from the environment by encapsulation within a defined compartment. Our aim is to model a hypothetical example of this system, using a nucleic acid-replicating enzyme encapsulated within a liposome. In addition to protocellular modelling, we expect our system to be applicable in biotechnology.
Two aspects of the system are significant to successfully obtain the production of nucleic acid within the liposomes. These are the encapsulation of the enzyme and the permeability of the membrane to the negatively charged nucleotide monomers.
The encapsulation method used is a dehydration/rehydration procedure. This method is mild, as no extreme fluctuations of temperature or any chemicals such as detergents or organic solvents that might negatively effect the activity of the enzyme are used (Shew and Deamer, 1983).
Significant ionic permeability has been shown by Paula and colleagues to exist only in liposomes made of lipids with hydrophobic chains 14 to 18 carbons long. This diffusion of ions across the lipid membrane is facilitated by transmembrane defects that allow ions to bypass the hydrophobic energy barrier of the bilayer (Paula et al., 1996). The contribution of transient defects to the permeability has been maximized in our laboratory by keeping the system at the phase transition temperature, where less stable regions exist at the boundaries between the two lipid phases, the gel and crystalline liquid states.
Chakrabarti and colleagues demonstrated that the template-independent enzyme poly-nucleotide phosphorylase can produce poly-adenylic acid within dimyrystoyl-sn-glycero-3-phosphocholine (DMPC) liposomes (Chakrabarti et al., 1994). DMPC is a saturated 14-carbon chain lipid, the shortest chain length that still results in stable liposomes. Maintaining the liposomes at the phase transition temperature of DMPC, 23°C, provided a permeability to adenosine diphosphate (ADP) sufficient for the enzyme to build a chain of the monomers.
Our current work is to produce a specific RNA sequence within a liposome by using an encapsulated template-directed enzyme, T7 RNA polymerase. This permeability of the membrane must be high enough for the enzyme to be supplied with specific nucleotides at the rate of about 200 nucleotides a second. This is higher than what was needed for the template-independent enzyme. The permeability in the Poly-nucleotide phosphorylase system was 2.6 X 10-10 cm/s; what we need is about 10-8 cm/s. We have made progress toward this by using thermal cycling, fluctuating the system above and below the phase transition temperature to maximize the occurance of transient defects. We have achieved rates of approximately 10-9 cm/s.
Results gathered in preliminary trials seemed to indicate that RNA was formed within some liposomes; these are photos of liposomes dyed with a fluorescent dye specific to nucleic acid. Further substantiation, in the form of a second detection method for the RNA product, is needed.
This project is supported by the National Aeronautics and Space Administration (NASA).
Chakrabarti, A., Breaker, R.R., Joyce, G.F. and Deamer, D. (1994) J. Mol. Evol., 39, 555-559. Production of RNA by a polymerase protein encapsulated within phospholipid vesicles
Paula, S., Volkov, A.G., Van Hoek, A.N., Haines, T.H. and Deamer, D.W. (1996) Biophys. J., 70, 339-348. Permeation of protons, potassium ions and small polar molecules through phospholipid bilayers as a function of membrane thickness
Shew, R. and Deamer, S. (1983) Biochim. Biophys. Acta, 816, 1-8. A novel method for encapsulating macromolecules in liposomes
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