BRET-BASED, DUAL PROTEASE BIOSENSOR

1 . A bioluminescence resonance energy transfer (BRET) sensor comprising a single polypeptide chain comprising:

1) a bioluminescent protein, or a mutant thereof;

2) a first BRET acceptor sequence comprising a monomeric NeonGreen (mNG) fluorescent protein; and

3) a second BRET acceptor sequence comprising a red fluorescent protein (RFP);

wherein the sensor detects activity of two proteins and wherein a first protein cleaves the first acceptor producing a change in green emission and the second protein cleaves the second acceptor producing a change in red emission.

2 . The sensor of claim 1 , wherein the bioluminescent protein is a luciferase, or an aequorin.

3 . The sensor of claim 1 , wherein the bioluminescent protein is selected from Renilla luciferase, firefly luciferase, and Gaussia luciferase, or a mutant thereof.

4 . The sensor of claim 1 , wherein the bioluminescent protein is selected from a nano-luciferase (NLuc), or a pic-luciferase (picALuc).

5 . The sensor of claim 1 , wherein the mNG fluorescent protein is attached to the N-terminal of the bioluminescent protein.

6 . The sensor of claim 1 , wherein the RFP is attached to the C-terminal of the bioluminescent protein.

7 . The sensor of claim 1 , wherein the RFP is selected from CyOFP1, mScarlet, LSS-mKate2, or mBeRFP tagged protein.

8 . The sensor of claim 1 , wherein:

(i) the bioluminescent protein has an emission spectrum with a peak emission;

(ii) the mNG fluorescent protein has a first excitation spectrum that overlaps with the emission spectrum; and

(iii) the RFP has a second excitation spectrum that overlaps with the emission spectrum, wherein the first excitation spectrum is separated from the second excitation spectrum.

9 . The sensor of claim 8 , wherein the second excitation spectrum is separated from the first excitation spectrum by about 10 nm to about 100 nm.

10 . The sensor of claim 8 , wherein the second excitation spectrum is separated from the first excitation spectrum by about 10 nm to about 50 nm.

11 . The sensor of claim 8 , wherein the second excitation spectrum is separated from the first excitation spectrum by about 10 nm to about 30 nm.

12 . The sensor of claim 8 , wherein the second excitation spectrum is separated from the first excitation spectrum by about 10 nm.

13 . The sensor of claim 1 , wherein the sensor detects proteolytic cleavage by two proteases.

14 . The sensor of claim 13 , wherein the sensor detects proteolytic cleavage of a coronavirus main protease (M pro ) and a coronavirus papain-like protease (PL pro ).

15 . The sensor of claim 1 , wherein the mNG fluorescent protein is a mNG coronavirus M pro protease, and the RFP is an RFP coronavirus PL pro protease.

16 . The sensor of either one of claim 14 or 15 , wherein the coronavirus is SARS-CoV-2.

17 . A method for detecting coronavirus infection in a patient, the method comprising

(i) contacting a biological sample from the patient with the sensor of claim 15 ;

(ii) adding a substrate for the bioluminescent protein to the mixture; and

(iii) measuring fluorescence in a green channel and a red channel;

wherein

a reduction in the green channel fluorescence of the sensor indicates the presence of the coronavirus M pro protease;

a reduction in the red channel fluorescence of the sensor indicates the presence of the coronavirus PL pro protease; and

wherein the detection of coronavirus M pro protease or coronavirus PL pro protease, or both, indicates that the patient has SARS-CoV-2 infection.

18 . A method for simultaneously testing inhibitors of coronavirus main protease (M pro ) and coronavirus papain-like protease (PL pro ), the method comprising

(i) contacting a test compound with a mixture comprising a coronavirus M pro and a coronavirus PL pro ;

(ii) adding a dual protease sensor of claim 15 to the mixture;

(iii) adding a substrate for the bioluminescent protein to the mixture; and

(iii) measuring fluorescence in a green channel and a red channel.

19 . The method of claim 18 , wherein the coronavirus is SARS-CoV-2.

20 . The method of claim 18 , wherein activity of a coronavirus M pro protease results in the cleavage of the sensor resulting in a decrease in the BRET between the bioluminescent protein and the mNG protein, thereby reducing the green fluorescence of the sensor, and indicating the presence of the coronavirus M pro protease; and

wherein if the compound inhibits the coronavirus M pro protease, the green fluorescence of the sensor will not be reduced.

21 . The method of claim 18 , wherein activity of a coronavirus PL pro protease results in the cleavage of the sensor resulting in a decrease of BRET between the bioluminescent protein and the RFP, thereby reducing the red fluorescence of the sensor, and indicating the presence of the coronavirus PL pro protease; and

wherein if the compound inhibits the coronavirus PL pro protease, the red fluorescence of the sensor will not be reduced.

22 . A dual protease sensor for detecting a coronavirus comprising an amino acid construct of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6.

23 . A plasmid comprising a nucleotide construct of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

24 . A cell transfected with a plasmid of claim 20 .

25 . A kit comprising the sensor of claim 1 , and a substrate for the bioluminescent protein.