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. An RNA construct comprising a non-naturally occurring, continuous sequence of ribonucleotide bases defining:
a stem-loop structure;
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, wherein the target polynucleotide is not continuous with the RNA construct;
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 base pair with complementary bases on the target polynucleotide spaced apart by an intermediate region on the target polynucleotide defining another three way junction joining region, whereby,
in the presence of the target polynucleotide, a stem-loop conformation of said construct is stabilized.
2. The construct 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 conformation.
3. The construct of claim 1 wherein the RNA construct further comprises RNA defining a polypeptide coding region and wherein
in the absence of the target polynucleotide, the construct assumes a conformation inhibiting association with an RNA binding protein and suppressing translation of the polypeptide-coding region, and
in the presence of the target polynucleotide, the construct assumes a stem-loop conformation promoting association with the RNA binding protein and promoting translation of the polypeptide coding region.
4. The construct of claim 3 wherein the polypeptide coding region is disposed 5′ of the first region.
5. The construct of claim 1 , wherein the target polynucleotide is characteristic of a pathogen.
6. The construct of claim 5 wherein the pathogen is a virus or a single-celled microorganism.
7. The construct of claim 1 , wherein the target polynucleotide is expressed preferentially in a cell type of a multicellular organism.
8. The construct of claim 7 wherein the cell type is a neoplastic cell.
9. The construct of claim 1 , wherein the target polynucleotide comprises a synthetic target polynucleotide or ssDNA for transfection into a cell.
10. A DNA vector comprising a transcription unit encoding a construct as in claim 1 .
11. A cell comprising a construct as in claim 1 .
12. The construct of claim 1 , wherein a joining region comprises no more than about 10 nucleotides.
13. The construct of claim 3 , wherein the polypeptide coding region encodes a polypeptide needed for survival of a cell whereby only in the presence of the construct and the target polynucleotide in the cell can the cell survive.
14. The construct of claim 3 , wherein the polypeptide coding region is expressed in a cell and encodes a polypeptide lethal to the cell.
15. The construct of claim 14 wherein the cell is an infected cell or a neoplastic cell, and the target polynucleotide is expressed by an organism infecting the cell or by the neoplastic cell.
16. The construct of claim 3 , wherein the polypeptide coding region encodes a detectable marker polypeptide whose detection indicates the presence of an analyte comprising the target polynucleotide.
17. The construct of claim 1 , wherein the construct comprises more than one said stem-loop structure.