First and second generation turn-on protease biosensors. a) Helical wheel diagram depicting the A/B coiled-coil, with sites for mutations shown in orange. b) TEV protease cleavage dependent change in luciferase activity in the singly inhibited A/B coiled-coil split-luciferase sensor. The translated proteins were incubated in the presence or absence of TEV (40 pmols) for 3 hrs and luminescence was measured. AB’/B refers to the B-NFluc and CFluc-ATEV-B’ sensor, AB’_2A/B refers to the B-NFluc and CFluc-A-TEV-B’_2A sensor and AB’_4A/B refers to the B-NFluc and CFluc-A-TEV-B’_4A sensor. c) SDS-PAGE analysis of the TEV protease cleaved translation products using ³⁵S methionine after 3 hrs. d) Helical wheel diagram depicting the EE/RR coiled-coil with sites for mutations shown in orange. e) TEV protease cleavage dependent change in luciferase activity in the singly inhibited EE/RR coiled-coil split reporter. The translated proteins were incubated in the presence or absence of TEV (40 pmols) for 3 hours and luminescence was measured. ER’/R refers to the RR-NFluc and CFlucEE-TEV-RR’ sensor, EERR’_3A/RR refers to the RR-NFluc and CFluc-EE-TEV-RR’_3A sensor, and EERR’_6A/B refers to the RR-NFluc and CFluc-EE-TEV-RR’_6A sensor. Please see text for details of the design.

Third generation doubly inhibited protease biosensors and application to caspase-3. a) Schematic representation of the doubly inhibited luciferase sensor, Helices A and A’ have identical amino acid sequences and have been labelled differently for clarity. b) TEV protease dependent change in luminescence signal in the doubly inhibited coiled coil-split reporter system. AB’/A’B, AB’_2A/A’B and AB’_4A/A’B refer to co-translations of the A’-TEV-B-NFluc half with either CFluc-A-TEV-B’, Cfluc-A-TEV-B’_2A, or CFluc-A-TEV-B’_4A, to yield the three different sensors. c) Caspase-3 dependent activation of doubly inhibited firefly luciferase split reporter system in the presence and absence of added caspases-3. AB’/A’B(CASP3) refers to the CFluc-A-CASP3-B’ and A’-CASP3-B-NFluc containing sensor.

Turn-on protease biosensor design: auto-inhibited intramolecular coiled-coil cleavage dependent split-protein complementation. CFluc; C-terminal fragment of firefly luciferase (Fluc), residues 398–550, NFluc; N-terminal fragment of Fluc, residues 2–416. Helices A and B’ comprise an intramolecular dimeric coiled-coil connected by a tobacco etch virus (TEV)-protease cleavage site. In the first generation design the coiled-coils, B and B’ have identical amino acid sequences. Upon incubation of the bipartite split-protein sensor (CFluc-A-TEV-B’ and B-NFluc) with TEV-protease, the linker is cleaved and the two firefly luciferase halves can potentially reassemble firefly luciferase that catalyzes the mono-oxygenation of luciferin to produce light.

Turn-on Protease Biosensor utilizing Split-β-Lactamase. a) Schematic presentation of the protease biosensor design based upon the singly inhibited β-lactamase split-protein reporter with either no (B’) or four mutations (B’_4A), b) Rate of hydrolysis of the Fluorocillin Green, β-Lactamase substrate, (Invitrogen) subsequent to the addition of TEV as a function of time. AB’/B refers to assays performed with co-translation of NβLac-A-TEV-B and B-CβLac, AB’_4A/B refers to co-translation of NβLac-A-TEV-B_4A and B-CβLac. c) The slope of the hydrolysis data from (b) represented as a bar graph for the indicated split-β-lactamase sensors in the presence and absence of TEV protease for comparison to the split-luciferase derived sensors.

Design of AND logic gates utilizing dual protease bionsensors. a) Protease specific activation of the TEV and caspase-3 turn-on biosensors. For the split-luciferase TEV protease biosensor, mRNA corresponding to A’-TEV-B-NFluc was co-translated with CFluc-A-TEV-B’ and either TEV or Caspase-3 was added. For the split-luciferase caspase-3 protease biosensor, mRNA corresponding to A’-CASP3-B-NFluc was co-translated with CFluc-A-CASP3-B’ and either caspase-3 or TEV was added and luminescence recorded. b) Dual protease biosensors; mRNA corresponding to either A’-TEV-B-NFluc and CFluc-A-CASP3-B’ or A’-CASP3-B-NFluc and CFluc-A-TEV-B’ were co-translated and appropriate proteases and buffers were added separately or together and luminescence recorded. The biosensors are fully activated only upon the addition of both TEV and caspase-3. A truth table for the AND logic gate (inset), wherein, an output of 1 is obtained only when both inputs equal 1.