Multiple input biologic classifier circuits for cells
Provided herein are high-input detector modules and multi-input biological classifier circuits and systems that integrate sophisticated sensing, information processing, and actuation in living cells and permit new directions in basic biology, biotechnology and medicine. The multi-input biological classifier circuits described herein comprise synthetic, scaleable transcriptional/post-transcriptional regulatory circuits that are designed to interrogate the status of a cell by simultaneously sensing expression levels of multiple endogenous inputs, such as microRNAs. The classifier circuits then compute whether to trigger a desired output or response if the expression levels match a pre-determined profile of interest.
1. A high-input detector module for classifying a cell status based on detecting whether an input microRNA is expressed at a specific level or higher than a reference level, said high-input detector module comprising:
(a) an inducible promoter sequence operably linked to:
(i) a repressor sequence encoding a repressor product, and
(ii) one or more target sequences for microRNA, said one or more target sequences for microRNA are the target sequences of the one or more input microRNAs the module is designed to detect;
(b) a repressible promoter sequence operably linked to an output sequence encoding an output product, said repressor product being specific for the repressible promoter sequence; and
(c) one or more regulatory units, comprising a constitutive or inducible promoter sequence operably linked to:
(i) a sequence encoding a transcriptional activator product, said transcriptional activator product activates the inducible promoter sequence operably linked to the repressor sequence and the one or more target sequences for microRNA of the module, and
(ii) one or more target sequences for microRNA, said one or more target sequences for microRNA are the target sequences of the one or more input microRNAs the module is designed to detect.
2. The high-input detector module of claim 1 , wherein said transcriptional activator product activates the inducible promoter sequence operably linked to:
(i) the sequence encoding the transcriptional activator product, and
(ii) the one or more target sequences for microRNA of the one or more regulatory units.
3. A multiple-input biological classifier circuit for classifying a cell status based on detecting in parallel an expression pattern of a subset of at least two different input microRNAs, each of which is expressed at a specific level or higher than a reference level, the biological classifier circuit comprising at least two high-input detector modules according to claim 1 .
4. The multiple-input biological classifier circuit of claim 3 , wherein the output product is a reporter protein, a transcriptional activator, a transcriptional repressor, a pro-apoptotic protein, a lytic protein, an enzyme, a cytokine, or a cell-surface receptor.
5. The multiple-input biological classifier circuit of claim 3 , wherein the repressor sequence of at least one of the high-input detector module further comprises a sequence encoding for a protein or agent that is a functional or physiological inhibitor of the output product of the multiple-input biological classifier circuit.
6. A pharmaceutical composition comprising the high-input detector module of claim 1 and a pharmaceutically acceptable compound.
7. A pharmaceutical composition comprising the multiple-input biological classifier circuit of claim 3 and a pharmaceutically acceptable compound.
8. A method for identifying a cell or population of cells in vitro, ex vivo, or in vivo based on an expression pattern of at least three different input microRNAs, the method comprising introducing the multiple-input biological classifier circuit of claim 3 into a cell or population of cells, wherein expression of an output product by the cell identifies the cell or population of cells.
9. A method for identifying a cell or population of cells in vitro, ex vivo, or in vivo based on an expression pattern of at least three different input microRNAs, the method comprising introducing the multiple-input biological classifier circuit of claim 3 into a cell or population of cells, wherein expression of an output product by the cell identifies the cell or population of cells.
10. A multiple-input biological classifier circuit for classifying a cell status based on detecting in parallel an expression pattern of a subset of at least two different input microRNAs, the biological classifier circuit comprising:
at least two types of input detector modules detecting expression of at least two different input microRNAs, at least one of the at least two different input microRNAs having a lower expression level than a reference expression level, and at least one of the at least two different input microRNAs having a higher expression level than the reference expression level, and
(a) one of the at least two input detector modules is a low-input detector module for detecting the at least one input microRNA expressed at a lower level than the reference expression level, said low-input detector module comprises a repressible promoter sequence operably linked to:
(i) an output sequence encoding an output product, and
(ii) at least one target sequence for microRNA specific for the at least one input microRNA having a lower expression level than the reference expression level; and
(b) one of the at least two input detector modules is a high-input detector module for detecting the at least one input microRNA expressed at a higher level than the reference expression level, said high-input detector module comprises a constitutive or inducible promoter sequence operably linked to:
(i) a repressor sequence that encodes for a repressor product, said repressor product represses the repressible promoter of the low input detector module, and
(ii) one or more target sequences for microRNA specific for the one or more input microRNAs having a higher expression level than the reference expression level;
wherein each of the at least one microRNA target sequences of each low-input detector module are different from each of the at least one microRNA target sequences of each high-input detector module; and
wherein expression of the output product classifies the cell status.
11. The multiple-input biological classifier circuit of claim 10 , wherein the constitutive or inducible promoter sequence of the at least one high-input detector module is an inducible promoter sequence.
12. The multiple-input biological classifier circuit of claim 11 , wherein the at least one of the high-input detector modules further comprises one or more regulatory units, the one or more regulatory units comprising a constitutive or inducible promoter sequence operably linked to:
(i) a sequence encoding a transcriptional activator product, said transcriptional activator product activates the inducible promoter sequence operably linked to the repressor sequence and the one or more target sequences for microRNA of the high-input module, and
(ii) the one or more target sequences for microRNA.
13. The multiple-input biological classifier circuit of claim 12 , wherein said transcriptional activator product activates the inducible promoter sequence operably linked to:
(i) the sequence encoding a transcriptional activator product, and
(ii) the one or more target sequences for microRNA of one or more regulatory units.
14. The multiple-input biological classifier circuit of claim 10 , wherein the repressor sequence of at least the one high-input detector module further comprises a sequence encoding a microRNA, wherein said microRNA is different from each different microRNA inputs detected by the circuit, and wherein the output sequence of the at least one low-input detector module further comprises a microRNA target sequence for the microRNA.
15. The multiple-input biological classifier circuit of claim 10 , wherein the output product is a reporter protein, a transcriptional activator, a transcriptional repressor, a pro-apoptotic protein, a lytic protein, an enzyme, a cytokine, or a cell-surface receptor.
16. The multiple-input biological classifier circuit of claim 10 , wherein the repressor sequence of the at least one high-input detector module further comprises a sequence encoding for a protein or agent that is a functional or physiological inhibitor of the output product of the multiple-input biological classifier circuit.
17. A pharmaceutical composition comprising the high-input detector module of claim 10 and a pharmaceutically acceptable compound.
18. A multiple-input biological classifier circuit for classifying a cell status based on detecting in parallel an expression pattern of a subset of at least three different input microRNAs, the biological classifier circuit comprising:
at least two types of input detector modules detecting expression of at least three different input microRNAs, at least one of the at least three different input microRNAs having a lower expression level than a reference expression level, at least one of the at least three different input microRNAs having a higher expression level than the reference expression level, and a third or more of the at least three different input microRNAs having a different expression level than the reference expression level, and:
(a) one of said at least two input detector modules is a low-input detector module for detecting each of the different input microRNAs expressed at a lower level than the reference expression level, said low-input detector module comprises a repressible promoter sequence operably linked to:
(i) an output sequence encoding an output product, and
(ii) one or more target sequences for microRNA specific for each of the different input microRNAs having a lower expression level than the reference expression level; and
(b) at least one of said at least two input detector modules is a high-input detector module for detecting one of the different input microRNAs expressed at a higher level than the reference expression level, wherein said high-input detector module comprises a promoter sequence operably linked to:
(i) a repressor sequence that encodes for a repressor product, said repressor product represses the repressible promoter of the low-input detector module, and
(ii) one or more target sequences for microRNA, said one or more microRNA target sequences are specific for the at least one of the different input microRNAs having a higher expression level than the reference expression level;
wherein each of the one or more microRNA target sequences of the low-input detector module is different from each microRNA target sequence of each high-input detector module; and
wherein expression of the output product classifies the cell status.
19. The multiple-input biological classifier circuit of claim 18 , wherein the constitutive or inducible promoter sequence of the at least on high-input detector module is an inducible promoter sequence.
20. The multiple-input biological classifier circuit of claim 19 , wherein the at least one of the high-input detector modules further comprises one or more regulatory units, the one or more regulatory units comprising a constitutive or inducible promoter sequence operably linked to:
(i) a sequence encoding a transcriptional activator product, said transcriptional activator product activates the inducible promoter sequence operably linked to the repressor sequence and the one or more target sequences for microRNA of the high-input module, and
(ii) the one or more target sequences for microRNA.
21. The multiple-input biological classifier circuit of claim 20 , wherein said transcriptional activator product activates the inducible promoter sequence operably linked to:
(i) the sequence encoding a transcriptional activator product, and
(ii) the one or more target sequences for microRNA of one or more regulatory units.