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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
First Claim
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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, andii) 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.

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