Phosphorylation-based miRNA sensor
AssigneeMassachusetts Institute of Technology
InventorsRon Weiss, Ross D. Jones, Jin Huh
Life of this patent
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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).
Claims18
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.
Dependencies are inferred from the claim text.