Compositions and methods for logic-gated profiling of biologic activity

A noninvasive method for determining protease activity in a biological sample from a subject, comprising:

• contacting the biological sample with a protease activity sensor comprising a first protease substrate and a second protease substrate, wherein the first and second protease substrates comprise protease cleavage sites,
• wherein the first substrate is cleaved by a first protease and the second substrate is cleaved by a second protease wherein the first protease and the second protease are different proteases, and
• wherein the protease activity sensor provides a detectable signal via a reporter molecule only when both the first protease substrate and the second protease substrate are cleaved;
• wherein the reporter molecule is released by the protease cleavage and diffuses into circulation and into tissues that can be obtained noninvasively,
• detecting the presence of the detectable signal in a noninvasively obtained sample;
• and
• identifying the activity of the first protease and second protease based on the presence of the detectable signal in the sample, wherein the first and second proteases comprise granzyme B (GzmB), thrombin (Thrb), a metalloproteinase, or a viral protease;
• wherein the protease activity sensor comprises SEQ ID NO:
• 1 or SEQ ID NO:
• 2.