Nanocircles

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.

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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. The randomized sequence acts as a promoter that allow initiation with RNA polymerase to transcribe the nanocircle.

Figure 1B. Schematic of artificial ribozymes using error prone reverse transcripase PCR

Results

Effect of Ligation

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.

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Figure 2. "Improvement of transcription activity over successive rounds of in vitro selection. RNA amount was measured for each successive population at 37°C after 1.5 h. Dark and light bars correspond to the relative RNA amounts (>80-nt product) for the successive population with and without ligation, respectively" (Olmichi et al. 2002).

Production of Monomeric Ribozymes

Three sets of randomized domains (E1, E15, and E38) were chosen to measure the total amount of RNA and the amount of monomeric RNA. All three domains produced more total RNA than either the initial library or a nanocircle lacking the randomized domain altogether (Figure 2A). The molecular size designated as 103nt indicates the amount of RNA that was self-processed into the smallest monomer. Thus, even though all three domains produced significant total amounts of DNA (Figure 3B), the amount of monomeric RNA of 103nt length differed greatly between the groups such that E15 produced the most, followed by E1, and lastly E38, which hardly produced any monomeric ribozymes in comparison (Figure 3C and 3A).

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Figure 2. "Selected circular DNA motifs engender RNA synthesis in vitro with E. coli RNAP. (A) Autoradiogram of denaturing 10% polyacrylamide gel showing in vitro transcription of the 103-nt initial library, a control 63-nt nanocircle lacking the randomized domain, and selected individual nanocircles E1, E15, and E38 (after 1.5 h). (B) The relative total RNA amounts (all lengths >80 nt) for the 103-nt initial library, 63-nt nanocircle lacking the randomized domain, and E1, E15, and E38. (C) Time course of the production of monomeric ribozyme for the 103-nt initial library, 63-nt nanocircle lacking the randomized domain, E1, and E15" (Ohmichi 2002).

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Figure 4. The domain E15 was used to apply to a different ribozyme portion known as marA to test whether various ribozymes can be constructed using the same promoter-like sequence. These results suggest that not only does the marA construct produce as much RNA and as much monomeric RNA as the hammerhead ribozyme but it in fact produces more RNA.

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Continuing Research

Artificial human telomerase Synthetic DNA nanocircles act as essentially infinite catalytic templates for efficient synthesis of long telomeres by DNA polymerase enzymes.

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