Caged sensors, regulators and compounds and uses thereof

• US 7,541,193 B2
• Filed: 11/17/2003
• Issued: 06/02/2009
• Est. Priority Date: 11/18/2002
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Panomics, Inc.
• Inventors: Nguyen, Quan, McMaster, Gary
• Primary Examiner(s): Nguyen; Bao-Thuy L
• Assistant Examiner(s): YU, MELANIE J
• Application Number:
  US10/716,174
• Publication Number:
  US 20040166553A1
• Time in Patent Office: 2,024 Days
• Field of Search: 435/4, 435/7.1, 435/7.2, 435/6, 435/7.6, 435/21, 435/15, 435/183, 435/287.1, 435/7.71, 435/7.72, 435/7.9, 435/174, 435/176, 435/177, 436/501, 436/518, 436/519, 436/86
• US Class Current: 436/172
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2525/207 siRNA, miRNA
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

A number of caged (e.g., photoactivatable) compositions are provided, for example, caged enzyme sensors, caged binding sensors, caged nucleic acid probes, caged interfering RNAs, caged antisense nucleic acids, caged ribozymes, caged biomolecular analogs, caged transcription factors, caged antibodies, caged molecular decoys, and caged aptamers. Compositions comprising a plurality of caged components are also provided, as are labeled and optionally caged modulators. Also provided are kits for making the caged compositions and methods for making and for using the caged compositions. Related systems and devices are also provided (e.g., an uncaging device that can trigger photoactivatable assays and optionally detect signals from the assays).

Claims

1. A composition, comprising:

a cell comprising an enzyme and a caged sensor for detecting an activity of the enzyme, which caged sensor comprises;

a) one or more molecules collectively comprising;

i) a substrate for the enzyme, wherein the substrate is in a first state on which the enzyme can act, thereby converting the substrate to a second state, and

ii) a first label, wherein a first signal exhibited by the first label when the substrate is in its first state is distinguishable from a second signal exhibited by the first label when the substrate is in its second state;

b) one or more first caging groups associated with the one or more molecules, the first caging groups inhibiting the enzyme from acting upon the substrate; and

,

c) optionally, one or more second caging groups associated with a phosphobinder;

wherein the enzyme is a protein kinase that phosphorylates tyrosine, serine and/or threonine;

wherein the one or more molecules comprise one polypeptide;

wherein the one polypeptide comprises the substrate for the kinase and the first label and comprises a second label or a quencher;

wherein the first label and the second label or the quencher interact to produce the first signal when the substrate is not phosphorylated; and

wherein phosphorylation of the substrate prevents the interaction of the first label and the second label or the quencher, thereby resulting in production of the second signal;

wherein phosphorylation of the substrate triggers a conformational change in the polypeptide, the conformational change preventing the interaction of the first label and the second label or the quencher;

or wherein phosphorylation of the substrate results in binding of the phosphobinder to the phosphorylated substrate, the binding of the phosphobinder preventing the interaction of the first label and the second label or the quencher;

wherein the presence of the second caging groups prevents the phosphobinder from binding the phosphorylated substrate.

3. The composition of claim 1 or 2, wherein the second caging groups are removable under different conditions than the first caging groups preventing phosphorylation of the substrate.

2. A composition, comprising:

an enzyme and a caged sensor for detecting an activity of the enzyme, which caged sensor comprises;

a) one or more molecules collectively comprising;

i) a substrate for the enzyme, wherein the substrate is in a first state on which the enzyme can act, thereby converting the substrate to a second state, wherein the first state is not converted to the second state by cleavage by the enzyme, and

ii) a first label, wherein a first signal exhibited by the first label when the substrate is in its first state is distinguishable from a second signal exhibited by the first label when the substrate is in its second state;

b) one or more first caging groups associated with the one or more molecules, the first caging groups inhibiting the enzyme from acting upon the substrate; and

,

c) optionally, one or more second caging groups associated with a phosphobinder, wherein the presence of the second caging groups prevents the phosphobinder from binding the phosphorylated substrate;

the first caging groups inhibiting the enzyme from acting upon the substrate;

wherein the enzyme is a protein kinase that phosphorylates tyrosine, seine and/or threonine;

wherein the one or more molecules comprise one polypeptide;

wherein the one polypeptide comprises the substrate for the kinase and the first label and comprises a second label or a quencher;

wherein the first label and the second label or the quencher interact to produce the first signal when the substrate is not phosphorylated; and

wherein phosphorylation of the substrate prevents the interaction of the first label and the second label or the quencher, thereby resulting in production of the second signal;

wherein phosphorylation of the substrate triggers a conformational change in the polypeptide, the conformational change preventing the interaction of the first label and the second label or the quencher;

or wherein phosphorylation of the substrate results in binding of the phosphobinder to the phosphorylated substrate, the binding of the phosphobinder preventing the interaction of the first label and the second label or the quencher.

4. A composition, comprising:

a cell comprising an enzyme and a caged sensor for detecting an activity of the enzyme, which caged sensor comprises;

a) one or more molecules collectively comprising;

i) a substrate for the enzyme, wherein the substrate is in a first state on which the enzyme can act, thereby converting the substrate to a second state, and

ii) a first label, wherein a first signal exhibited by the first label when the substrate is in its first state is distinguishable from a second signal exhibited by the first label when the substrate is in its second state;

b) one or more first caging groups associated with the one or more molecules, the first caging groups inhibiting the enzyme from acting upon the substrate; and

,

c) optionally, a phosphobinder comprising an antibody, an SH-2 domain, a PTB domain, a 14-3-3 domain, an FHA domain, a WD40 domain and/or a WW domain;

wherein the enzyme is a protein kinase that phosphorylates tyrosine, serine and/or threonine;

wherein the one or more molecules comprise one polypeptide;

wherein the one polypeptide comprises the substrate for the kinase and the first label and comprises a second label or a quencher;

wherein the first label and the second label or the quencher interact to produce the first signal when the substrate is not phosphorylated; and

wherein phosphorylation of the substrate prevents the interaction of the first label and the second label or the quencher, thereby resulting in production of the second signal;

wherein phosphorylation of the substrate triggers a conformational change in the polypeptide, the conformational change preventing the interaction of the first label and the second label or the quencher;

or wherein phosphorylation of the substrate results in binding of the phosphobinder to the phosphorylated substrate, the binding of the phosphobinder preventing the interaction of the first label and the second label or the quencher.

5. A composition, comprising:

a cell comprising an enzyme and a caged sensor for detecting an activity of the enzyme, which caged sensor comprises;

a) one or more molecules collectively comprising;

i) a substrate for the enzyme, wherein the substrate is in a first state on which the enzyme can act, thereby converting the substrate to a second state, and

ii) a first label, wherein a first signal exhibited by the first label when the substrate is in its first state is distinguishable from a second signal exhibited by the first label when the substrate is in its second state;

b) one or more first caging groups associated with the one or more molecules, the first caging groups inhibiting the enzyme from acting upon the substrate; and

,

c) one or more second caging groups associated with a phosphobinder, wherein the presence of the second caging groups prevents the phosphobinder from binding the phosphorylated substrate;

wherein the enzyme is a protein kinase that phosphorylates tyrosine, seine and/or threonine;

wherein the one or more molecules comprise one polypeptide;

wherein the one polypeptide comprises the substrate for the kinase and the first label;

wherein the polypeptide comprises the phosphobinder and a second label or a quencher;

wherein the first label and the second label or the quencher do not interact when the substrate is not phosphorylated, thereby producing the first signal; and

wherein phosphorylation of the substrate results in intramolecular binding of the phosphobinder to the phosphorylated substrate, the intramolecular binding resulting in the interaction of the first label and the second label or the quencher, thereby producing the second signal;

or, wherein the one or more molecules comprise a first polypeptide and a second polypeptide;

wherein the first polypeptide comprises the substrate for the kinase and the first label, wherein the second polypeptide comprises the phosphobinder and a second label or a quencher;

wherein the first label and the second label or the quencher do not interact when the substrate is not phosphorylated, thereby producing the first signal; and

wherein phosphorylation of the substrate results in intermolecular binding of the phosphobinder to the phosphorylated substrate, the intermolecular binding resulting in the interaction of the first label and the second label or the quencher, thereby producing the second signal.

7. The composition of claim 5 or 6, wherein the second caging groups are removable under different conditions than the first caging groups preventing phosphorylation of the substrate.

6. A composition, comprising:

an enzyme and a caged sensor for detecting an activity of the enzyme, which caged sensor comprises;

a) one or more molecules collectively comprising;

i) a substrate for the enzyme, wherein the substrate is in a first state on which the enzyme can act, thereby converting the substrate to a second state, wherein the first state is not converted to the second state by cleavage by the enzyme, and

ii) a first label, wherein a first signal exhibited by the first label when the substrate is in its first state is distinguishable from a second signal exhibited by the first label when the substrate is in its second state;

b) one or more first caging groups associated with the one or more molecules, the first caging groups inhibiting the enzyme from acting upon the substrate; and

,

c) one or more second caging groups associated with a phosphobinder, wherein the presence of the second caging groups prevents the phosphobinder from binding on the phosphorylated substrate;

wherein the enzyme is a protein kinase that phosphorylates tyrosine, serine and/or threonine;

wherein the one or more molecules comprise one polypeptide;

wherein the one polypeptide comprises the substrate for the kinase and the first label;

wherein the polypeptide comprises the phosphobinder and a second label or a quencher;

wherein the first label and the second label or the quencher do not interact when the substrate is not phosphorylated, thereby producing the first signal; and

wherein phosphorylation of the substrate results in intramolecular binding of the phosphobinder to the phosphorylated substrate, the intramolecular binding resulting in the interaction of the first label and the second label or the quencher, thereby producing the second signal;

or, wherein the one or more molecules comprise a first polypeptide and a second polypeptide;

wherein the first polypeptide comprises the substrate for the kinase and the first label;

wherein the second polypeptide comprises the phosphobinder and a second label or a quencher;

wherein the first label and the second label or the quencher do not interact when the substrate is not phosphorylated, thereby producing the first signal; and

wherein phosphorylation of the substrate results in intermolecular binding of the phosphobinder to the phosphorylated substrate, the intermolecular binding resulting in the interaction of the first label and the second label or the quencher, thereby producing the second signal.

8. A composition, comprising:

a cell comprising an enzyme and a caged sensor for detecting an activity of the enzyme, which caged sensor comprises;

a) one or more molecules collectively comprising;

i) a substrate for the enzyme, wherein the substrate is in a first state on which the enzyme can act, thereby converting the substrate to a second state, and

ii) a first label, wherein a first signal exhibited by the first label when the substrate is in its first state is distinguishable from a second signal exhibited by the first label when the substrate is in its second state;

b) one or more first caging groups associated with the one or more molecules, the first caging groups inhibiting the enzyme from acting upon the substrate; and

,

c) a phosphobinder comprising;

an antibody, an SH-2 domain, a PTB domain, a 14-3-3 domain, an FHA domain, a WD40 domain and/or a WW domain;

wherein the enzyme is a protein kinase that phosphorylates tyrosine, seine and/or threonine;

wherein the one or more molecules comprise one polypeptide;

wherein the one polypeptide comprises the substrate for the kinase and the first label;

wherein the polypeptide comprises the phosphobinder and a second label or a quencher;

wherein the first label and the second label or the quencher do not interact when the substrate is not phosphoryiated, thereby producing the first signal; and

wherein phosphorylation of the substrate results in intramolecular binding of the phosphobinder to the phosphorylated substrate, the intramolecular binding resulting in the interaction of the first label and the second label or the quencher, thereby producing the second signal;

or, wherein the one or more molecules comprise a first polypeptide and a second polypeptide;

wherein the first polypeptide comprises the substrate for the kinase and the first label;

wherein the second polypeptide comprises the phosphobinder and a second label or a quencher;

wherein the first label and the second label or the quencher do not interact when the substrate is not phosphorylated, thereby producing the first signal; and

wherein phosphorylation of the substrate results in intermolecular binding of the phosphobinder to the phosphorylated substrate, the intermolecular binding resulting in the interaction of the first label and the second label or the quencher, thereby producing the second signal.

9. A composition, comprising:

a cell comprising an enzyme and a caged sensor for detecting an activity of the enzyme, which caged sensor comprises;

a) one or more molecules collectively comprising;

i) a substrate for the enzyme, wherein the substrate is in a first state on which the enzyme can act, thereby converting the substrate to a second state, and

ii) a first label, wherein a first signal exhibited by the first label when the substrate is in its first state is distinguishable from a second signal exhibited by the first label when the substrate is in its second state; and

,

b) one or more first caging groups associated with the one or more molecules, the first caging groups inhibiting the enzyme from acting upon the substrate;

wherein the enzyme is a protein kinase that phosphorylates tyrosine, serine and/or threonine;

wherein the one or more molecules comprise one polypeptide;

wherein the one polypeptide comprises the substrate for the kinase, a second substrate, the first label, a third label, a fourth label or a quencher, and a phosphobinder;

the substrate comprising a seine, threonine, or tyrosine residue capable of being phosphorylated by the kinase;

the second substrate being associated with one or more third caging groups, the presence of which prevents phosphorylation of the second substrate;

wherein the first label is located at the seine, threonine, or tyrosine residue and exhibits the first signal when the residue is not phosphorylated and the second signal when the residue is phosphorylated;

wherein the third label and the fourth label or the quencher do not interact when the second substrate is not phosphorylated, thereby producing a third signal; and

wherein phosphorylation of the second substrate results in intramolecular binding of the phosphobinder to the phosphorylated second substrate, the intramolecular binding resulting in the interaction of the third label and the fourth label or the quencher, thereby producing a fourth signal, the fourth signal distinguishable from the first, second and third signals.

10. A composition, comprising:

an enzyme and a caged sensor for detecting an activity of the enzyme, which caged sensor comprises;

a) one or more molecules collectively comprising;

i) a substrate for the enzyme, wherein the substrate is in a first state on which the enzyme can act, thereby converting the substrate to a second state, wherein the first state is not converted to the second state by cleavage by the enzyme, and

ii) a first label, wherein a first signal exhibited by the first label when the substrate is in its first state is distinguishable from a second signal exhibited by the first label when the substrate is in its second state; and

,

b) one or more first caging groups associated with the one or more molecules, the first caging groups inhibiting the enzyme from acting upon the substrate;

wherein the enzyme is a protein kinase that phosphorylates tyrosine, seine and/or threonine;

wherein the one or more molecules comprise one polypeptide;

wherein the one polypeptide comprises the substrate for the kinase, a second substrate, the first label, a third label, a fourth label or a quencher, and a phosphobinder;

the substrate comprising a seine, threonine, or tyrosine residue capable of being phosphorylated by the kinase;

the second substrate being associated with one or more third caging groups, the presence of which prevents phosphorylation of the second substrate;

wherein the first label is located at the seine, threonine, or tyrosine residue and exhibits the first signal when the residue is not phosphorylated and the second signal when the residue is phosphorylated;

wherein the third label and the fourth label or the quencher do not interact when the second substrate is not phosphorylated, thereby producing a third signal; and

wherein phosphorylation of the second substrate results in intramolecular binding of the phosphobinder to the phosphorylated second substrate, the intramolecular binding resulting in the interaction of the third label and the fourth label or the quencher, thereby producing a fourth signal, the fourth signal distinguishable from the first, second and third signals.

11. The composition of claim 9 or 10, wherein the second substrate is for the same kinase or for a different kinase.

12. The composition of claim 9 or 10, wherein the one or more third caging groups are located on a residue that can be phosphorylated by the kinase.

13. The composition of claim 12, wherein the third caging groups preventing phosphorylation of the second substrate are removable under different conditions than the first caging groups preventing phosphorylation of the substrate.

14. The composition of claim 9 or 10, wherein one of the third label and the fourth label or the quencher is located at the C-terminus of the polypeptide and the other of the third label and the fourth label or the quencher is within the polypeptide.

15. The composition of claim 9 or 10, wherein the third and fourth labels are fluorophores capable of exhibiting FRET.

16. The composition of claim 9 or 10, wherein the phosphobinder is associated with one or more second caging groups, the presence of which prevents the phosphobinder from binding the phosphorylated second substrate.

17. The composition of claim 16, wherein the second caging groups are removable under different conditions than the first caging groups preventing phosphorylation of the substrate and/or under different conditions than the third caging groups preventing phosphorylation of the second substrate.

18. The composition of claim 9 or 10, wherein the phosphobinder comprises an antibody, an SH-2 domain, a PTB domain, a 14-3-3 domain, an FHA domain, a WD40 domain and/or a WW domain.

19. A composition, comprising:

a cell comprising an enzyme and a caged sensor for detecting an activity of the enzyme, which caged sensor comprises;

a) one or more molecules collectively comprising;

i) a substrate for the enzyme, wherein the substrate is in a first state on which the enzyme can act, thereby converting the substrate to a second state, and

ii) a first label, wherein a first signal exhibited by the first label when the substrate is in its first state is distinguishable from a second signal exhibited by the first label when the substrate is in its second state, and,

b) one or more first caging groups associated with the one or more molecules, the first caging groups inhibiting the enzyme from acting upon the substrate;

wherein the one or more molecules comprise a fifth label, the fifth label exhibiting a unique fifth signal, the fifth signal being independent of the state of the substrate.

21. The composition of claim 19 or 20, wherein the fifth label is a fluorophore or a quantum dot.

20. A composition, comprising:

an enzyme and a caged sensor for detecting an activity of the enzyme, which caged sensor comprises;

a) one or more molecules collectively comprising;

i) a substrate for the enzyme, wherein the substrate is in a first state on which the enzyme can act, thereby converting the substrate to a second state, wherein the first state is not converted to the second state by cleavage by the enzyme, and

ii) a first label, wherein a first signal exhibited by the first label when the substrate is in its first state is distinguishable from a second signal exhibited by the first label when the substrate is in its second state, and,

b) one or more first caging groups associated with the one or more molecules, the first caging groups inhibiting the enzyme from acting upon the substrate;

wherein the one or more molecules comprise a fifth label, the fifth label exhibiting a unique fifth signal, the fifth signal being independent of the state of the substrate.

22. A composition, comprising:

an enzyme and a caged sensor for detecting an activity of the enzyme, which caged sensor comprises;

a) one or more molecules collectively comprising;

i) a substrate for the enzyme, wherein the substrate is in a first state on which the enzyme can act, thereby converting the substrate to a second state, wherein the first state is not converted to the second state by cleavage by the enzyme, and

ii) a first label, wherein a first signal exhibited by the first label when the substrate is in its first state is distinguishable from a second signal exhibited by the first label when the substrate is in its second state;

b) one or more first caging groups associated with the one or more molecules, the first caging groups inhibiting the enzyme from acting upon the substrate; and

,

c) optionally, a phosphobinder comprising;

an antibody, an SH-2 domain, a PTB domain, a 14-3-3 domain, an FHA domain, a WD40 domain and/or a WW domain;

wherein the enzyme is a protein kinase that phosphorylates tyrosine, seine and/or threonine;

wherein the one or more molecules comprise one polypeptide;

wherein the one polypeptide comprises the substrate for the kinase and the first label and comprises a second label or a quencher;

wherein the first label and the second label or the quencher interact to produce the first signal when the substrate is not phosphorylated; and

wherein phosphorylation of the substrate prevents the interaction of the first label and the second label or the quencher, thereby resulting in production of the second signal;

wherein phosphorylation of the substrate triggers a conformational change in the polypeptide, the conformational change preventing the interaction of the first label and the second label or the quencher;

or wherein phosphorylation of the substrate results in binding of the phosphobinder to the phosphorylated substrate, the binding of the phosphobinder preventing the interaction of the first label and the second label or the quencher.

23. A composition, comprising:

an enzyme and a caged sensor for detecting an activity of the enzyme, which caged sensor comprises;

a) one or more molecules collectively comprising;

i) a substrate for the enzyme, wherein the substrate is in a first state on which the enzyme can act, thereby converting the substrate to a second state, wherein the first state is not converted to the second state by cleavage by the enzyme, and

ii) a first label, wherein a first signal exhibited by the first label when the substrate is in its first state is distinguishable from a second signal exhibited by the first label when the substrate is in its second state; and

,

b) one or more first caging groups associated with the one or more molecules, the first caging groups inhibiting the enzyme from acting upon the substrate;

wherein the enzyme is a protein kinase that phosphorylates tyrosine, seine and/or threonine;

wherein the one or more molecules comprise one polypeptide;

wherein the one polypeptide comprises the substrate for the kinase and the first label;

wherein the polypeptide comprises a phosphobinder and a second label or a quencher;

wherein the first label and the second label or the quencher do not interact when the substrate is not phosphorylated, thereby producing the first signal; and

wherein phosphorylation of the substrate results in intramolecular binding of the phosphobinder to the phosphorylated substrate, the intramolecular binding resulting in the interaction of the first label and the second label or the quencher, thereby producing the second signal;

or, wherein the one or more molecules comprise a first polypeptide and a second polypeptide;

wherein the first polypeptide comprises the substrate for the kinase and the first label;

wherein the second polypeptide comprises a phosphobinder and a second label or a quencher;

wherein the first label and the second label or the quencher do not interact when the substrate is not phosphorylated, thereby producing the first signal; and

wherein phosphorylation of the substrate results in intermolecular binding of the phosphobinder to the phosphorylated substrate, the intermolecular binding resulting in the interaction of the first label and the second label or the quencher, thereby producing the second signal;

wherein the phosphobinder comprises an antibody, an SH-2 domain, a PTB domain, a 14-3-3 domain, an FHA domain, a WD40 domain and/or a WW domain.