Difference between revisions of "Nanocircles"
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Rolling circle transcription produces identical ribozyme sequences that can then self-process, or cleave themselves into monomers, and form their secondary structures. Then, the monomeric ribozymes are reverse transcribed into [http://en.wikipedia.org/wiki/Complementary_DNA cDNA] in the process of mutagenic PCR. A biotin tag on the RNA strand allows for the complementary strands to be separated by using streptavidin magnetic beads and denaturing the strands. To recreate a nanocircle, the resulting DNA is bound at the ends with a short strand of DNA that acts as a splint so that when T4 ligase is added, the DNA is already arranged in a circle so that the ligase can bind the beginning and end of the ssDNA. | Rolling circle transcription produces identical ribozyme sequences that can then self-process, or cleave themselves into monomers, and form their secondary structures. Then, the monomeric ribozymes are reverse transcribed into [http://en.wikipedia.org/wiki/Complementary_DNA cDNA] in the process of mutagenic PCR. A biotin tag on the RNA strand allows for the complementary strands to be separated by using streptavidin magnetic beads and denaturing the strands. To recreate a nanocircle, the resulting DNA is bound at the ends with a short strand of DNA that acts as a splint so that when T4 ligase is added, the DNA is already arranged in a circle so that the ligase can bind the beginning and end of the ssDNA. | ||
http://www.pnas.org/content/vol0/issue2001/images/data/012589099/DC1/5890Fig9.gif | http://www.pnas.org/content/vol0/issue2001/images/data/012589099/DC1/5890Fig9.gif | ||
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| + | Figure 1A. Structrure of single-stranded DNA nanocircle composed of 63 nucleotides encoding a hammerhead ribozyme and 41 nucleotides of randomized sequences | ||
Figure 1B. Schematic of artificial ribozymes using error prone reverse transcripase PCR | Figure 1B. Schematic of artificial ribozymes using error prone reverse transcripase PCR | ||
Revision as of 09:38, 20 November 2007
Nanocircles are small circular single-stranded DNA that can be transcribed by phage and bacterial RNA polymerases. These plasmid-like structures were originally developed by Eric T. Kool's lab. The new technology uses a method called rolling circle transcription (RCT) to encode hammerhead, hairpin and hepatitis delta ribozymes.
Rolling Circle Animation Click on Rolling Circles & Artificial Telomeres
Goals
- Synthesize efficient self-processing ribozymes
- Regulatation of genes using ribozymes
- Change ribozymes while retaining randomized domain to emphasize universality
- Interchange genes for utility
- Reinforce importance of secondary structure in cleaving properties
Experimental Design
Rolling circle transcription produces identical ribozyme sequences that can then self-process, or cleave themselves into monomers, and form their secondary structures. Then, the monomeric ribozymes are reverse transcribed into cDNA in the process of mutagenic PCR. A biotin tag on the RNA strand allows for the complementary strands to be separated by using streptavidin magnetic beads and denaturing the strands. To recreate a nanocircle, the resulting DNA is bound at the ends with a short strand of DNA that acts as a splint so that when T4 ligase is added, the DNA is already arranged in a circle so that the ligase can bind the beginning and end of the ssDNA.
http://www.pnas.org/content/vol0/issue2001/images/data/012589099/DC1/5890Fig9.gif
Figure 1A. Structrure of single-stranded DNA nanocircle composed of 63 nucleotides encoding a hammerhead ribozyme and 41 nucleotides of randomized sequences
Figure 1B. Schematic of artificial ribozymes using error prone reverse transcripase PCR
Results
http://www.pnas.org/content/vol99/issue1/images/medium/pq0125890002.gif
Figure 3. Rolling circle transcription can produce much more RNA than can transcription of linear, unligated DNA. Ligation is essential for RCT because it allows for the nanocircles that are best able to produce the most RNA to amplify these selective advantages to subsequent generations.
http://www.pnas.org/content/vol99/issue1/images/medium/pq0125890004.gif http://www.pnas.org/content/vol99/issue1/images/medium/pq0125890006.gif
http://www.pnas.org/content/vol99/issue1/images/medium/pq0125890007.gif
http://www.pnas.org/content/vol99/issue1/images/medium/pq0125890008.gif
Applications of Ribozymes in Synthetic Systems - Danielle Jordan
