Trans-acting RNA switches
Disclosed are RNA constructs which function to activate or inactivate a biological process, e.g., may be designed for attachment to a polypeptide coding region. Such RNA constructs modulate translation of a polypeptide from the coding region in response to the presence of a target polynucleotide in an expression environment. Such RNA constructs include a weakened stem-loop structure which, when bound to the target polynucleotide, assumes stem-loop secondary structure and associates with an RNA binding protein. Association with the RNA binding protein modulates translation of the polypeptide coding region. Such RNA constructs also have three-way junction joining regions 3′ and 5′ of the stem-loop structure.
1. A method for modulating translation of a polypeptide coding region comprising the step of:
(A) providing a translatable RNA construct comprising a polypeptide coding region ligated to a non-naturally occurring RNA comprising a continuous sequence of ribonucleotide bases, the continuous sequence of ribonucleotide bases defining:
a stem-loop structure which associates with an RNA binding protein present in an expression environment to modulate translation of the polypeptide coding region when the RNA construct is in a stem-loop conformation;
simulated, three way junction joining regions 3′ and 5′ of the stem-loop structure;
a first region 5′ of the 5′ joining region comprising bases complementary to a 3′ region of a target polynucleotide;
a second region 3′ of the 3′ joining region comprising bases complementary to a 5′ region of the target polynucleotide;
the base sequence of the first and second regions being selected to hybridize with complementary bases on the target polynucleotide spaced apart by an intermediate region on the target polynucleotide defining another three way junction joining region; and
(B) introducing the translatable RNA construct into an expression system, whereby,
in the absence of the target polynucleotide, the construct assumes a conformation inhibiting association with the RNA binding protein, and
in the presence of the target polynucleotide, the construct assumes a stem-loop conformation promoting association with the RNA binding protein.
2. The method of claim 1 , wherein at least one base in the stem of the stem-loop structure is mismatched with its potential binding partner so as to reduce the stability of the stem-loop structure.
3. The method of claim 1 , wherein a joining region comprises no more than about 10 nucleotides.
4. The method of claim 1 , wherein step (B) is effected by transfecting a DNA encoding the translatable RNA construct.
5. The method of claim 1 , wherein the target polynucleotide is characteristic of a pathogen.
6. The method of claim 1 , wherein the target polynucleotide is expressed preferentially in a cell type of a multicellular organism.
7. The method of claim 6 , wherein the cell type is a neoplastic cell.
8. The method of claim 1 , comprising the additional step of introducing the target polynucleotide into the expression environment.
9. The method of claim 1 , wherein the expression environment is a cell and the polypeptide coding region encodes a polypeptide lethal to the cell.
10. The method of claim 9 , wherein the cell is an infected cell or a neoplastic cell, and the target polynucleotide is expressed by the neoplastic cell or by an organism infecting the cell.
11. The method of claim 1 , wherein the polypeptide coding region comprises a sequence encoding an enzyme or pore-forming protein.
12. The method of claim 1 , wherein the polypeptide coding region encodes a detectable marker.
13. The method of claim 12 , wherein the detectable marker is fluorescent.
14. The method of claim 1 , wherein translation of the coding region is suppressed when the RNA binding protein binds to the stem-loop structure.
15. The method of claim 1 , wherein translation of the coding region is promoted when the RNA binding protein binds to the stem-loop structure.
16. The method of claim 1 , wherein the construct comprises more than one stem-loop structure.