Part:BBa_K2624004
U6-inhSi-pri-miRNA
The corresponding short transcript of RNA would base-pair with the basal segments of the designed pri-miRNA analogue so that the pri-miRNAs cannot be recognized and processed to the corresponding pre-miRNAs. But it also contains a RNA promoter for the RNA-dependent RNA polymerase NS5B. Upon substitutional hybridation catalyzed by the polymerase, the inhibitory strand unbinds with the pri-miRNA so that the RNA interference takes effect.
Please refer to the wiki of CPU_CHINA 2018 because this part is strictly related to the other parts used in the project. You may need it if you follow their conditional RNA interference strategy in your study.
Usage
The 2018 CPU_CHINA project is a gene therapy strategy based on conditional RNA interference. The RNAi is conditional because the pri-miRNA analogue (BBa_K2624005) taking effect is blocked by this inhibitory strand. But then it can be opened since we introduced a RNA promoter, by the corresponding RNA-dependent RNA polymerase, if the transfected cell is cancerous.
We could only imagine this part to be used under a similar circumstance where you want to block a pri-miRNA from getting processed by the Drosha-DGCR8 complex.
Biology
The U6 promoter is a RNA Polymerase III promoter. Pol III genes do not get polyadenylated and thus are ideal for making small nuclear RNAs such as shRNA hairpins. Pol II transcription requires a poly-A site to terminate the mRNA and process it correctly, which is not what we want in the inhibitory strand.
It is the single-stranded nature of the basal segments, rather than the nucleotide sequences, that may be critical for Drosha processing of pri-miRNAs. Like Han et al., we block the basal segments by treating the pri-miRNA with an oligonucleotide that is complementary to these segments. The oligonucleotide was in fact designed to bind to both the 5’ and 3’ basal segments simultaneously and specifically repress the cleavage of the pri-miRNA we designed (BBa_K2624005).
An RNA composed of a stem-loop and a single-stranded sequence is commonly used to initiate viral RNA synthesis. The Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is able to direct de novo (oligonucleotide primer-independent) synthesis. And a 25-nucleotide (nt) RNA, termed SLD3, was found by Kao et al. to be capable of supporting efficient RNA synthesis by NS5B. In our case SLD3 is just after the inhibitory strand, so that recombinant NS5B in the nucleus is able to release the active pri-miRNA analogue by substitutional hybridation.
The Hepatitis D Virus ribozyme forms a specific tertiary RNA conformation that triggers self-cleavage immediately at the SLD3 RdRP Promoter border。
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 312
Illegal NgoMIV site found at 341 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 305
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