Dynamic control of colocalization of proteins

Patent metadata

Field	Value
Publication number	US-11530278-B2
Application number	US-202016751793-A
Country	US
Kind code	B2
Filing date	Jan 24, 2020
Priority date	Jan 24, 2019
Publication date	Dec 20, 2022
Grant date	Dec 20, 2022

Abstract

The present invention provides a method for controlling colocalization of two or more proteins in a cell. The method comprises expressing the proteins, scaffold RNA molecules having binding motifs for the proteins, and a trigger RNA molecule in the cell. In the presence of the trigger RNA molecule, a scaffold may be assembled (ON) by the scaffold RNA molecules via hybridization such that the proteins may be colocalized; or disassembled (OFF) such that the proteins may be separated and not colocalized. The proteins may provide a biological activity when colocalized or not colocalized.

First claim

What is claimed:

1. A method for controlling colocalization of a first heterologous protein with a second heterologous protein in a cell, comprising:
(a) expressing the first heterologous protein and a first scaffold RNA molecule in the cell, wherein the first scaffold RNA molecule comprises a first binding motif, a hybridization sequence and a toehold sequence, the first heterologous protein is bound to the first binding motif, and the hybridization sequence is bound to a first sequence complementary with the hybridization sequence;
(b) expressing the second heterologous protein and a second scaffold RNA molecule in the cell, wherein
the second scaffold RNA molecule comprises a second binding motif and the first sequence complementary with the hybridization sequence, the second heterologous protein is bound to the second binding motif, whereby the first heterologous protein is colocalized with the second heterologous protein in the cell;

(c) expressing a trigger RNA molecule in the cell, wherein the trigger RNA molecule comprises a first trigger sequence complementary with the toehold sequence and a second trigger sequence complementary with the hybridization sequence,
(d) binding the first trigger sequence complementary with the toehold sequence to the toehold sequence and binding the second trigger sequence complementary with the hybridization sequence to the hybridization sequence whereby the first sequence complementary with the hybridization sequence is separated from the hybridization sequence and the first heterologous protein is not colocalized with the second heterologous protein in the cell.

2. The method of claim 1 , wherein the first heterologous protein and the second heterologous protein are different, and the first binding motif and the second binding motif are different.

3. The method of claim 2 , wherein the first heterologous protein and the second heterologous protein are expressed under the same promoter.

4. The method of claim 1 , wherein each of the first binding motif and the second binding motif has a hairpin sequence.

5. The method of claim 4 , wherein the hairpin sequence consists of 25-35 nucleotides.

6. The method of claim 4 , wherein the hairpin sequence consists of 30 nucleotides.

7. The method of claim 1 , wherein the cell is selected from the group consisting of E. coli, S. cerevisiae , and HeLa cells.

8. The method of claim 1 , further comprising expressing a combined scaffold RNA molecule in the cell, and cleaving the combined scaffold RNA to generate the first scaffold RNA molecule and the second scaffold RNA molecule in the cell.

9. The method of claim 1 , further comprising expressing a third heterologous protein and a third scaffold RNA molecule in the cell, wherein the third scaffold RNA molecule comprises a third binding motif and a third sequence consisting of a polynucleotide sequence identical to that of the hybridization sequence, and the third heterologous protein is bound to the third binding motif, whereby the first sequence complementary with the hybridization sequence is bound to the third sequence consisting of a polynucleotide sequence identical to that of the hybridization sequence, and the second heterologous protein is colocalized with the third heterologous protein.

10. A method for controlling colocalization of a first heterologous protein with a second heterologous protein in a cell, comprising:
(a) expressing the first heterologous protein and a first scaffold RNA molecule in the cell, wherein the first scaffold RNA molecule comprises a first binding motif, a hybridization sequence, a first sequence complementary with the hybridization sequence and a toehold sequence, the first heterologous protein is bound to the first binding motif, and the hybridization sequence is bound to the first sequence complementary with the hybridization sequence;
(b) expressing the second heterologous protein and a second scaffold RNA molecule in the cell, wherein the second scaffold RNA molecule comprises a second binding motif and a second sequence consisting of a polynucleotide sequence identical to the hybridization sequence, the second heterologous protein is bound to the second binding motif, whereby the first heterologous protein is not colocalized with the second heterologous protein in the cell;
(c) expressing a trigger RNA molecule in the cell, wherein the trigger RNA molecule comprises a first trigger sequence complementary with the toehold sequence and a second trigger sequence complementary with the hybridization sequence,
(d) binding the first trigger sequence complementary with the toehold sequence to the toehold sequence, whereby the first sequence complementary with the hybridization sequence is separated from the hybridization sequence, and
(e) binding the second trigger sequence complementary with the hybridization sequence to the hybridization sequence, whereby the second sequence consisting of a polynucleotide sequence identical to that of the hybridization sequence is bound to the first sequence complementary with the hybridization sequence, and the first heterologous protein is colocalized with the second heterologous protein in the cell.

11. The method of claim 1 , wherein the first heterologous protein and the second heterologous protein provide a biological activity in the cell when the first heterologous protein is colocalized with the second heterologous protein, and the biological activity is reduced when the first heterologous protein is not colocalized with the second heterologous protein.

12. The method of claim 1 , wherein the first heterologous protein and the second heterologous protein provide a biological activity in the cell when the first heterologous protein is not colocalized with the second heterologous protein, and the biological activity is reduced when the first heterologous protein is colocalized with the second heterologous protein.

13. The method of claim 1 , wherein the first heterologous protein is a first fusion protein of a first enzyme and a first binding protein capable of binding to the first binding motif, and the second heterologous protein is a second fusion protein of a second enzyme and a second binding protein capable of binding to the second binding motif, wherein the first enzyme and the second enzyme provide a biological activity in the cell when the first heterologous protein is colocalized with the second heterologous protein, and the biological activity is reduced when the first heterologous protein is not colocalized with the second heterologous protein.

14. The method of claim 1 , wherein the first heterologous protein is a first fusion protein of a first enzyme and a first binding protein capable of binding to the first binding motif, and the second heterologous protein is a second fusion protein of a second enzyme and a second binding protein capable of binding to the second binding motif, wherein the first enzyme and the second enzyme provide a biological activity in the cell when the first heterologous protein is not colocalized with the second heterologous protein, and the biological activity is reduced when the first heterologous protein is colocalized with the second heterologous protein.

15. The method of claim 13 , wherein each of the first binding protein and the second binding protein is selected from the group consisting of Csy4 from P. aeruginosa , Cse3 from E. coli , Cse3 from T. thermophilus , and Cas6 from P. furiosus.

16. The method of claim 13 , wherein the cell produces a metabolite, and wherein the production of the metabolite by the cell is increased when the first hetero

Assignees

• Chen Wilfred
• Mitkas Alexander A
• Univ Delaware

Inventors

• Chen Wilfred
• Mitkas Alexander A

Classifications

• C07D209/18

  Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals · CPC title

• C12N15/111

  General methods applicable to biologically active non-coding nucleic acids · CPC title

• C07C39/19

  containing carbon-to-carbon double bonds but no carbon-to-carbon triple bonds · CPC title

• C07D403/14

  containing three or more hetero rings · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title