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| Status |
Public on Jun 20, 2024 |
| Title |
Specific silencing of pathogenic mRNA by a novel compact RNA-targeting tool TaqTth-hpRNA [Amplicon sequencing] |
| Organism |
Escherichia coli BL21 |
| Experiment type |
Other
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| Summary |
Pathogenic allele silencing is a promising treatment for genetic hereditary diseases. However, the concern about the specificity of present RNA-knockdown strategies has limited their in vivo applications. Here a TaqTth-hpRNA system consisting of a small, chimeric protein (TaqTth) and hairpin-RNA probe (hpRNA) is provided. The TaqTth-hpRNA showed a high-specific knockdown against targeted mRNA with minimal flanking sequence-motif requirement and less cell viability damage, then was applied to mutant APPswe mRNA silencing without altering the wild-type APP mRNA in Alzheimer’s disease. Notably, the combination of the TaqTth and human apolipoprotein E 2 (APOE2) overexpression encoded in a single AAV vector is available due to the compact size of TaqTth, and showed stronger inhibition of pathologies in the mouse model. Altogether, we provided the basis for a small RNA-targeting tool with high selectivity, minimal flanking sequence-motif requirement, less cell viability damage and potentially being an alternative in therapeutic applications.
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| Overall design |
Sequence preference detection. An oligo sequence, with two degeneracy bases at the 5' end, a target in the middle, and four degeneracy bases at the 3' end, was designed and inserted into the ampicillin start codon to construct a plasmid library. The ampicillin-resistant plasmid library, the chloramphenicol-resistant TaqTth protein expression plasmid and the spectinomycin-resistant probe plasmid were transferred into the electrical natural competence by bio-red electroporation. The expression of TaqTth was induced by the Tet inducer. Then 16 hours later, cells were scraped off plates and extracted plasmid DNA. The unrelated probe NT was used as the control group. To assess sequence preference, the nucleotide portion of the original library containing the target sequence was extracted and PCR-specific amplification was performed. The specific PCR product was sequenced by Sangon Biotech (Shanghai, China).
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| Contributor(s) |
Xu S |
| Citation(s) |
38972974 |
| BioProject |
PRJNA1121589 |
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| Submission date |
Jun 11, 2024 |
| Last update date |
Aug 08, 2024 |
| Contact name |
Shu Xu |
| E-mail(s) |
shuxu@cpu.edu.cn
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| Organization name |
China Pharmaceutical University
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| Street address |
China Pharmaceutical University
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| City |
Nanjing |
| State/province |
Jiangsu Province |
| ZIP/Postal code |
210009 |
| Country |
China |
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| Platforms (1) |
| GPL34578 |
Illumina HiSeq 4000 (Escherichia coli BL21) |
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| Samples (2) |
| GSM8322621 |
3-17NT_R1:Escherichia coli str. BL21:AMPLICON |
| GSM8322622 |
3-17T_R1:Escherichia coli str. BL21:AMPLICON |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSE269593_RAW.tar |
260.0 Kb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
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