Post-transcriptional regulation of RNA-related processes using encoded protein-binding RNA aptamers
In vitro SELEX has been used to discover high affinity RNA aptamers interacting with the tetracycline repressor protein in a tetracycline-dependent manner. Using in silico RNA folding predictions to guide the design of both aptamer truncations and mutants, minimized tetracycline repressor protein high affinity binding aptamers have been defined. Using one such aptamer, inducible post-transcriptional regulation in vivo has been demonstrated that is predicated on a direct interaction between a tetracycline repressor protein and a RNA aptamer element. These aptamer components can be integrated in any organism to inducibly regulate RNA translation of a gene of interest.
1. A method comprising:
a) providing:
i) a biological cell comprising a ribonucleic acid sequence encoding an open reading frame for a protein of interest;
ii) an aptamer sequence ligated within one hundred bases of a 5′ cap of said ribonucleic acid sequence, wherein said aptamer sequence comprises a tetracycline inducible protein binding site and a stem loop, wherein said binding site is in a loop portion of said stem loop, wherein said binding site comprises a first nucleotide sequence comprising the 5′-C/U-A/G/U-G/A-A/C/G-A-U/A-G/A-U/A-3′ consensus sequence and a second nucleotide sequence comprising the 5′-C/G/U-A/U/G-G-A-G-A-A-3′ consensus sequence;
iii) a tetracycline repressor protein comprising a tetracycline binding site; and
iv) a tetracycline-based compound, wherein said compound is capable of binding to said tetracycline binding site;
b) binding said tetracycline repressor protein to said protein binding site wherein translation of said protein of interest is reduced; and
c) contacting said tetracycline binding site with said tetracycline-based compound under conditions that relieves the translation reduction of said protein of interest.
2. The method of claim 1 , wherein said ribonucleic acid sequence further comprises a 5′ untranslated region.
3. The method of claim 1 , wherein said ribonucleic acid sequence further comprises a 3′ untranslated region.
4. The method of claim 1 , wherein said tetracycline-based compound is tetracycline.
5. The method of claim 1 , wherein said tetracycline-based compound is anhydrotetracycline.
6. The method of claim 1 , wherein said biological cell further comprises a plasmid configured to express said protein of interest.
7. The method of claim 1 , wherein said tetracycline repressor protein is a fusion protein.
8. The method of claim 7 , wherein said fusion protein comprises a translation initiation repressor protein.
9. The method of claim 7 , wherein said fusion protein comprises a polyadenylyl polymerase inhibitor protein domain.