Phosphorylation-based miRNA sensor
Provided herein are genetic circuits and cell state classifiers for detecting the microRNA profile of a cell. The cell state classifiers of the present disclosure utilize phosphorylation state of a transcription factor to control classifier output. Kinases and phosphatase pairs that function in phosphorylating or dephosphorylating the transcription factor are integrated into the circuit, their expression tuned by the presence of microRNAs of interest (e.g., in a cell). The genetic circuits and cell state classifiers may be used in various applications (e.g., therapeutic or diagnostic applications).
1. A cell state classifier, comprising:
(i) a first sensor circuit comprising a constitutive promoter operably linked to a nucleotide sequence encoding an activator, and a constitutive promoter operably linked to a nucleotide sequence encoding a kinase that phosphorylates the activator and produces a phosphorylated activator, and one or more target sites for a first microRNA;
(ii) a second sensor circuit comprising a constitutive promoter operably linked to a nucleotide sequence encoding a phosphatase that de-phosphorylates the phosphorylated activator, and one or more target sites for a second microRNA; and
(iii) a signal circuit comprising an activatable promoter that is activated by the phosphorylated activator, operably linked to a nucleotide sequence encoding an output molecule, and one or more target sites for the first microRNA,
optionally wherein the constitutive promoter of (i) and the constitutive promoter of (ii) are the same or wherein the constitutive promoter of (i) and the constitutive promoter of (ii) are different,
wherein the kinase, the phosphatase, and/or the activator are members of a bacterial two-component signaling system, wherein the bacterial two-component system comprises a histidine kinase comprising an amino acid sequence motif of HEXXN, HEXXT, or HDXXXP, wherein X is any amino acid, and a response regulator, and
wherein the phosphatase is a variant of the histidine kinase and comprises an amino acid substitution in the E or D of the HEXXN, HEXXT or HDXXXP motif.
2. The cell state classifier of claim 1 ,
wherein the kinase is a variant of the histidine kinase and comprises an amino acid substitution in the N, T, or P of the HEXXN, HEXXT or HDXXXP motif,
optionally wherein the kinase comprises an alanine substitution in the N, T, or P of the HEXXN, HEXXT, or HDXXXP motif.
3. The cell state classifier of claim 2 , wherein the histidine kinase is selected from the group consisting of: EnvZ, NarX, and PhoR,
optionally wherein the histidine kinase comprises the amino acid sequence of SEQ ID NO: 1, optionally wherein the phosphatase comprises an amino acid substitution corresponding to a D244A substitution in SEQ ID NO: 1, optionally wherein the phosphatase comprises the amino acid sequence of SEQ ID NO:2;
optionally wherein the histidine kinase comprises an amino acid substitution corresponding to a T247A substitution in SEQ ID NO: 1, optionally wherein the histidine kinase comprises the amino acid sequence of SEQ ID NO: 3;
optionally wherein the phosphatase comprises a dimerization and histidine phosphorylation (DHp) domain of EnvZ, optionally wherein the phosphatase comprises the amino acid sequence of SEQ ID NO: 4 or wherein the histidine kinase comprises two DHp domains fused to a cytoplasmic domain of EnvZ, optionally wherein the histidine kinase comprises the amino acid sequence of SEQ ID NO: 5.
4. The cell state classifier of claim 2 , wherein the activator comprises the response regulator of the bacterial two-component system; or wherein the activator comprises the response regulator of the bacterial two-component system fused to an activation domain, optionally wherein the activation domain is selected from the group consisting of: VP16, VP64, p65, and VPR; and/or
wherein the activation domain is selected from the group consisting of: VP16, VP64, p65, and VPR;
optionally wherein the response regulator is selected from the group consisting of: OmpR, NarL, NtrC, and PhoB.
5. The cell state classifier of claim 1 , wherein the phosphatase is a variant of the histidine kinase and comprises an alanine substitution in the E or D of the HEXXN, HEXXT, or HDXXXP motif.
6. The cell state classifier of claim 1 , wherein the activatable promoter comprises one or more response elements that binds to the activator;
optionally wherein the response element comprises one or more operators of the activator;
optionally wherein the activatable promoter further comprises a minimal promoter fused to the one or more response elements.
7. The cell state classifier of claim 1 , wherein the one or more target sites for the first microRNA is located upstream and/or downstream of the nucleotide sequence encoding the activator and the nucleotide sequence encoding the kinase in the first sensor circuit, optionally wherein 4 target sites for the first microRNA are located upstream and/or downstream of the nucleotide sequence encoding the activator and the nucleotide sequence encoding the kinase in the first sensor circuit; and/or
wherein the one or more target sites for the first microRNA is located upstream and/or downstream of the nucleotide sequence encoding the output molecule in the signal circuit; and/or
wherein 4 target sites for the first microRNA are located upstream and/or downstream of the nucleotide sequence encoding the output molecule in the signal circuit; and/or
wherein the one or more target sites for the second microRNA is located upstream and/or downstream of the nucleotide sequence encoding the phosphatase in the second sensor circuit, optionally wherein 4 target sites for the second microRNA are located upstream and/or downstream of the nucleotide sequence encoding the phosphatase in the second sensor circuit.
8. The cell state classifier of claim 1 , wherein the output molecule is a detectable molecule or a therapeutic molecule.
9. A method comprising delivering the cell state classifier of claim 1 to an in vitro or ex vivo cell and detecting an output molecule.
10. A method of treating a disease or disorder comprising delivering the cell state classifier of claim 1 to a cell, wherein the output molecule is a therapeutic molecule that is effective for treating the disease or disorder,
optionally wherein the cell is a diseased cell, or optionally wherein the cell is a cancer cell.
11. A method of diagnosing a disease or disorder comprising delivering the cell state classifier of claim 1 to an in vitro or ex vivo cell,
optionally wherein the cell is a diseased cell, or optionally wherein the cell is a cancer cell.
12. The method of claim 11 , the method further comprising detecting the output molecule,
optionally wherein the expression of the output molecule indicates the disease or disorder, or
optionally wherein the lack of expression of the output molecule indicates the disease or disorder.
13. A method of treating a disease or disorder, the method comprising administering an effective amount of a composition comprising the cell state classifier of claim 1 to a subject in need thereof, wherein the output molecule is a therapeutic molecule that is effective for treating the disease or disorder, optionally wherein the composition further comprises a pharmaceutically acceptable carrier.
14. A method of diagnosing a disease or disorder comprising administering an effective amount of a composition comprising the cell state classifier of claim 1 to in vitro or ex vivo cells of a subject in need thereof, and detecting the output molecule, optionally wherein the composition further comprises a pharmaceutically acceptable carrier.
15. An isolated cell comprising the cell state classifier of claim 1 ;
optionally wherein the cell is a prokaryotic cell, optionally a bacterial cell;
optionally wherein the cell is a eukaryotic cell, optionally a plant cell, an insect cell, or a mammalian cell, optionally a human cell.
16. The isolated cell of claim 15 , wherein the cell is a diseased cell, optionally a cancer cell.
17. The isolated cell of claim 15 , wherein the cell does not express the first microRNA; and/or
wherein the cell expresses the second microRNA; and/or
wherein the cell expresses the first microRNA and does not express the second microRNA; and/or
wherein the cell expresses the first microRNA and expresses the second microRNA; and/or
wherein the cell does not express the first microRNA and does not express the second microRNA.
18. A method comprising maintaining the cell of claim 15 in vitro or ex vivo, optionally further comprising detecting the output molecule, or optionally further comprising classifying the cell.