Phosphorylation-based mirna sensor

Abstract

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.   
     
     
         2 .- 3 . (canceled) 
     
     
         4 . The cell state classifier of  claim 1 , wherein the kinase, the phosphatase, and/or the activator are members of a bacterial two-component signaling system. 
     
     
         5 . The cell state classifier of  claim 4 , wherein bacterial two-component system comprises a histidine kinase comprises an amino acid sequence motif of HEXXN, HEXXT, or HDXXXP, wherein X is any amino acid, and a response regulator. 
     
     
         6 .- 20 . (canceled) 
     
     
         21 . The cell state classifier of  claim 5 , wherein the activator comprises the response regulator of the bacterial two-component system. 
     
     
         22 . The cell state classifier of  claim 5 , wherein the activator comprises the response regulator of the bacterial two-component system fused to an activator domain. 
     
     
         23 .- 25 . (canceled) 
     
     
         26 . The cell state classifier of  claim 1 , wherein the activatable promoter comprises one or more response elements that binds to the activator. 
     
     
         27 . (canceled) 
     
     
         28 . The cell state classifier of  claim 26 , wherein the activatable promoter further comprises a minimal promoter fused to the one or more response elements. 
     
     
         29 . 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. 
     
     
         30 . (canceled) 
     
     
         31 . 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 output molecule in the signal circuit. 
     
     
         32 . (canceled) 
     
     
         33 . The cell state classifier of  claim 1 , 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. 
     
     
         34 .- 36 . (canceled) 
     
     
         37 . A cell comprising the cell state classifier of  claim 1 . 
     
     
         38 .- 49 . 
     
     
         50 . A method comprising maintaining the cell of  claim 37 . 
     
     
         51 . The method of  claim 50 , further comprising detecting the output molecule. 
     
     
         52 . The method of  claim 50 , further comprising classifying the cell. 
     
     
         53 . A method comprising delivering the cell state classifier of  claim 1  to a cell and detecting an output molecule. 
     
     
         54 . 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. 
     
     
         55 .- 56 . (canceled) 
     
     
         57 . A method of diagnosing a disease or disorder comprising delivering the cell state classifier of  claim 1  to a cell. 
     
     
         58 .- 59 . (canceled) 
     
     
         59 . (canceled) 
     
     
         60 . The method of  claim 57 , the method further comprising detecting the output molecule. 
     
     
         61 .- 62 . (canceled) 
     
     
         63 . 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. 
     
     
         64 . 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 a subject in need thereof, and detecting the output molecule. 
     
     
         65 . (canceled)