| Summary: | 博士 === 國立中興大學 === 生物化學研究所 === 106 === -1 programed frameshifting (-1 PRF) leads to 5’ direction slippage of the ribosome during translational decoding. It is triggered by a stimulator RNA and a specific slippage site, and is important for efficient replication of a variety of viruses, including coronaviruses. Outbreak of the SARS-CoV in Asia in 2003 was followed by the emergence of MERS-CoV in Saudi Arabia and South Korea about ten years later. The virologist and the epidemiologist predict there will be more and more novel human coronaviruses emerging due to rapid mutation of viral genomes and the zoonotic features. Unfortunately, there are no FDA-approved vaccines for SARS and MERS. Therefore, the development of an antivirus strategy for rapid response to the emerging coronavirus infection is important.
In 2011, antisense peptide nucleic acids (PNAs) was used to target the conserved RNA sequence of SARS-CoV viral -1 PRF signal, and inhibited the replication of an SARS replicon. However, I found MERS-CoV -1 PRF might be composed by two stimulator RNA conformations: one is an H-type pseudoknot and the other is a kissing-hairpin type stimulator RNA. Such multiple stimulator conformations might hinder the design of antisense in targeting downstream viral -1 PRF stimulation. Here I demonstrated that an upstream duplex mediated by antisense 2’OMe-RNA attenuated -1 PRF efficiency of a reporter containing MERS viral sequence (p2luc MERSex) in human cell lysate. Furthermore, I also demonstrated its general -1 PRF attenuation activity toward distinct stimulator conformations. Thus, the upstream duplex also provides a strategy to quickly inhibit viral -1 PRF without the need of downstream stimulator information.
Aiming to apply this strategy to generating an antiviral stable cell line, I constructed a 293T stable cell line harboring a tetracycline inducible MERS targeting antisense RNA at the 3’UTR of mCherry. In order to trace this antisense RNA in live cell, I fused the antisense RNA with broccoli aptamer, which can interact with DFHBI-1T, a non-fluorescent dye, to emit green fluorescence. At the same time, the red fluorescence from mCherry can serve as the indication of this antisense RNA is being delivered into the cytoplasm. I successfully observed both red and green fluorescent signal in the stable cell-line, suggesting this approach can be used as a mehod for cytoplasmic functional RNA delivery in alternative to the RNA Pol I/III driven approach.
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