Applications of Ribozymes in Synthetic Systems - Danielle Jordan

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Contents

History and Background

What are ribozymes?

Ribozymes, also known as RNA enzymes or catalytic RNA, are RNA molecules that cataylze chemical reactions. They are able to catalzye hydrolysis of their own phosphodiester bonds or other RNA bonds. Some, such as RNA polymerase ribozymes, are able to catalyze their own synthesis. Wikipedia.

How do you make artificial ribozymes?

The method of directed evolution is used to create specific ribozymes. Large quantities of RNA are produced using polymerase enzymes. The large library of ribozymes are mutated and amplified using error prone rtPCR (reverse transcriptase PCR). One method of selection is by using biotin tags, which are covalently bonded to a particular substrate and can then be extracted by streptavidin-magnetic beads. Thus, the molecules that exhibit the optimal ligase activity are recoved using the streptavidin matrix. Wikipedia.

Why are they being used?

Current protein promoters systems cannot easily be transplanted from prokaryotic to eukaryotic organisms because the genomes and use of promoters are very differnt. However, ribozymes can be used in both systems because ribozymes do not rely on the cell's genetic information. Also, ribozymes can be artifically selected to respond to any set of exogenous molecules whereas there are only a limited number of protein promoters. Lastly, there are several mechanisms, such as by using nanocircles, by which ribozymes can be easily added to a cell without having to be integrated into the cell's existing DNA. One disadvantage is that ribozymes will not naturally stay permanently in a cell but rather be transported out of the cell or diluted over time. Wikipedia.

Ribozymes in Synthetic Systems

Nanocircles

Efficient bacterial transcription of DNA nanocircle vectors with optimized single-stranded promoters Ohmichi et al. 2001.

Supplementary Online Material

Ribozyme Switch

A modular and extensible RNA-based gene-regulatory platform for engineering cellular function Win and Smolke. 2007.

Supplementary Online Material

Ribozyme vesicles

RNA Catalysis in Model Protocell Vesicles Chen et al. 2005

Supplementary Online Material

References

Chen IA, Salehi-Ashtiani K, Szostak JW (2005). RNA Catalysis in Model Protocell Vesicles. Journal of the American Chemical Society 127(38):13213-9. Paper

Lindstrom UM, Chandrasekaran RA, Orbai L, Helquist SA, Miller GP, Oroudjev E, Hansma HG, Kool ET (2002). Artificial human telomeres from DNA nanocircle templates. PNAS 99(25):15953-8. Epub 2002 Nov 20. Abstract

Ohmichi T, Maki A, Kool ET (2001). Efficient bacterial transcription of DNA nanocircle vectors with optimized single-stranded promoters. PNAS 99(1):54-9. Epub 2001 Dec 18. Abstract.

Win MN, Smolke CD (2007). A modular and extensible RNA-based gene-regulatory platform for engineering cellular function. PNAS 104(36):14283-8. Epub 2007 Aug 20. Abstract.

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