Part:BBa_K3201002
S.mansoni Type 1 Hammerhead Ribozyme
The Type 3 Hammerhead Ribozyme (HHRz) originates from the helminth S.mansoni, and it is a self cleaving ribozyme.
Usage and Biology
This is the original Hammerhead ribozyme from S. mansoni. We have computationally modelled the molecule to determine its structure, which is shown in Figure 1.
We assessed its catalytic activity through an in vitro HHR cleavage assay. First, we produced sufficient amounts of the ribozyme through in vitro transcription. We incorporated a T7 promoter and used the in vitro transcription kit from NEB (T2050S). The in vitro transcription products were run in a 1% agarose gel and the result is shown in Figure 2.
Then we prepared the in vitro cleavage assay. In an eppendorf tube the following were added: 0.5 μL of in vitro transcribed HHR, 0.5 μL of 1 M Tris-HCl (final concentration 50 mM), 0.3 μL of 1 M MgCl2 (final concentration 30 mM), and 8.7 μL of nuclease-free water. The mixture was incubated in an incubator at 37 degrees Celsius for 1 hour. After 1 hour, the reaction was stopped using 10 μL of 6X loading dye. The mixture was then heated at 70 degrees Celsius for 5 minutes and then loaded on a gradient (4-20%) polyacrylamide gel. Before electrophoresis, the gel was prerun for 30 mins at 150V. The PAGE was done using 1X TBE as the running buffer and at a constant voltage of 150 V. The gel was stained overnight in Ethidium Bromide. The result is shown in Figure 3.
Successful cleavage results to the disappearance of a band at around 200 bp, which is the size of the HHR approximately (including its terminator).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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