Enhancing RNA catalysis through compartmentalization

Open Access
- Author:
- Molden, Rosalynn Conniff
- Graduate Program:
- Chemistry
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- July 17, 2009
- Committee Members:
- Christine Dolan Keating, Thesis Advisor/Co-Advisor
Christine Dolan Keating, Thesis Advisor/Co-Advisor - Keywords:
- model cell
aqueous two-phase systems
hammerhead ribozyme kinetics
RNA
cellular compartmentalization - Abstract:
- Experimental models of cellular conditions are useful because they can be used to study cell processes in a controlled environment and because they can be used to model prebiotic conditions. The influence of macromolecular crowding and compartmentalization on the cleavage rates of bimolecular minimal hammerhead ribozymes was investigated using aqueous two-phase systems (ATPS) as a model of the cell cytoplasm. The ATPS consisted of poly(ethyleneglycol) (PEG), dextran, 10 mM MgCl2 and buffer. The PEG-rich and dextran-rich phases of the ATPS segregate to create chemically distinct environments which cause differences in the local concentrations of RNA and provide a macromolecularly crowded environment similar to the cell cytoplasm. It was found that RNA concentrates to the dextran-rich phase of a PEG/dextran ATPS in a length-dependent manner and that long RNA sequences localize almost completely to the dextran-rich phase. It was also found that the local concentration of ribozyme in the dextran-rich phase of an ATPS could be controlled by changing the volume ratio of dextran-rich phase to PEG-rich phase. For most of the hammerhead ribozymes studied, increasing the local concentration of ribozyme also increased the observed rate of cleavage. There was a maximum of a 20-fold enhancement in the observed cleavage rate for 2 nM hammerhead ribozyme in a 250 µL ATPS with a 1:100 dextran to PEG-rich phase ratio compared to 2 nM hammerhead ribozyme in just dextran-rich phase. This study shows that compartmentalization can enhance RNA function, which may be important in RNA therapeutics, study of the RNA world, and further developing ATPS as a primitive model of the cell.