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.
Contents
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 1. (A) 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. (B) Schematic of artificial ribozymes using error prone reverse transcripase PCR. Image Permission Pending.
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). Image Permission Pending.
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 3. "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 et al., 2002). Image Permission Pending.
Specificity and Modularity of Ribozyme
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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Figure 4. "Assessment of transplantability of E15 selected motif to a new nanocircle encoding marA ribozyme. Autoradiogram of denaturing 10% polyacrylamide gel showing in vitro transcription of the 103-nt initial library, nanocircle E15, the new marA nanocircle, marA nanocircle with inactivated ribozyme, and two 63-nt nanocircle controls" (Ohmichi et al., 2002). Image Permission Pending.
Activity of Nanocircle Vector in E. coli
To test whether marA can be used in another system, the ribozyme was encoded in the upstream end of a CAT gene. When marA RNA is cleaved in trans, down-regulation of CAT activity would occur. Thus, not only was CAT-activity downregulated by the marA nanocircle vector, but it also showed a concentration dependence (Figure 5A and 5B). Image Permission Pending.
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Figure 5. "Effect of nanocircle vectors on the inhibition of CAT activity. (A) Thin-layer chromatogram showing levels of CAT expressed in the presence of 10 µM marA vector and E15 vector. The control lane is with no nanocircle vector. (B) Concentration dependence of down-regulation of CAT activity with marA vector" (Olmichi et al., 2002). Image Permission Pending.
Importance of Ribozyme Secondary Structure
The investigators used variations of marA that either lacked a trans cleavage point in the inactive marA or was missing a significant part of the marA in the short marA. This simply shows the only marA has a drastic decrease in % CAT activity, underscoring the importance of secondary structures in ribozymes. On the other hand, the inactive marA exhibited a signifant repression in the gene expression, which suggests that some of the ability that marA has in down-regulation of CAT may be attributed to anti-sense activity.
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Figure 6. "(A) Sequences and predicted secondary structures of the monomer ribozymes: active and inactive marA, and short marA. The inactivating A C mutation is boxed in the first ribozyme. (B) Effect of 10 µM various nanocircle vectors on the inhibition of CAT activity. The plotted data were averaged from three independent experiments" (Olmichi et al., 2002). Image Permission Pending.
Continuing Research
Artificial human telomerase Synthetic DNA nanocircles act as essentially infinite catalytic templates for efficient synthesis of long telomeres by DNA polymerase enzymes.
