TITLE: SARS-CoV-2 nsp3 macrodomain Time-Resolved FRET peptide displacement assay
AUTHORS: Haim Barr, Noa Lahav

[DESCRIPTION]
This protocol details the Time-Resolved FRET (TR-FRET) assay for SARS-CoV-2 nsp3 macrodomain (Mac1) binding of adenosine diphosphate (ADP)–ribosylated (ADPr) peptide. This method measures the affinity of Mac1 to its potential inhibitors by using a specific ADPr-modified peptide that allows the detection of binding. When bound, the biotinylated-peptide and the HIS-tagged Mac1 form a proximity complex that is detected by TR-FRET using Streptavidin-Eu Cryptate and anti-HIS-XL665 as a donor/acceptor pair. Excitation of the Eu Cryptate complex at 325 nm emits a resonant energy of 625 nm which in turn excites the XL665 to emit fluorescence at 665 nm. This energy transfer occurs only when ADPr modified peptide is in sufficient proximity to Mac1 while inhibitors which displace the peptide will prevent energy transfer. Binding is reported as the ratio of Acceptor/Donor (Em/Em) X 10,000.
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Experiment Concentrations (From Stock to Assay)
     ReagentStockLoaded into CombiFinal in assay plateUnitsHis-SARS COV2 MAC1183000 5012.5nM Substrate (Biotin-ADPr) 100000001600400nMDetection solution    Streptavidin-XL665 (SA-XL)10.250.125%MAb Anti-6HIS-Eu cryptate Gold1000.250.125%Assay buffer    HEPES pH=7.025025 25 mMNaCl200 20 20 mMBSA0.50.050.05%Tween 20 0.50.050.05%HTRF PPI Europium Detection Buffer1001010 %
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Compound Plate Design for Dose Response:
    Total Assay Volume: 16 µL
    Compounds Top Assay Concentration: 100 µM
    Dilution Factor: 3
    Dose Response Points: 10
    Number of Replicates: 2
    Backfill with DMSO: Yes

Compounds Plate Design for 2-Point Assay:
    Total Assay Volume: 16 µL
    Compounds Assay Concentration: 100 µM and 50 µM
    Dilution Factor: 2
    Dose Response Points: 2
    Number of Replicates: 2
    Backfill with DMSO: Yes
 

[BEFORE_START]
Note: Inhibitor compounds stock concentration is 20 millimolar (mM). Compounds are pre-dispensed into 384 plates and stored at -20˚C until use.

[STEPS]
SECTION: Prepare Reagents
2. PREPARE all of the reagents/buffers required for this experiment.

 Assay Buffer
 
 Reagents (dilute reagents in assay buffer for required volume)
 
Detection Solution (dilute reagents in assay buffer for required volume)
 

SECTION: Prepare 384-well Plate
3. PRIME Multi-Drop Combi Tube Dispensing Cassette with Assay Buffer by selecting the PRIME button on the Combi Dispenser until the tubes are filled completely. 
Note: Be sure to cycle dispensing several times on a clean plate lid (This confirms there are no bubbles in the Dispensing Cassette).
SECTION: Prepare 384-well Plate
3.1. DISPENSE 4 µL to Columns 1 and 23 of assay plate
Note: These will represent the inhibitor control columns

SECTION: Prepare 384-well Plate
3.2. EMPTY Multi-Drop Combi Tube Dispensing Cassette (by selecting the EMPTY button on the Combi Dispenser until the tubes of the cassette are emptied). Discard the Assay Buffer discharged from the cassette.
SECTION: Prepare 384-well Plate
4. PRIME Multi-Drop Combi Tube Dispensing Cassette with His-SARS COV2 MAC1 Enzyme by selecting the PRIME button on the Combi Dispenser until the tubes are filled completely. 
Note: Be sure to cycle dispensing several times on a clean plate lid (This confirms there are no bubbles in the Dispensing Cassette).
SECTION: Prepare 384-well Plate
4.1. DISPENSE 4 µL 50 nanomolar (nM) to Columns 2-22 and 24 of assay plate
Note: 
 50 nanomolar (nM) is four times the final concentration for the assay. It will be diluted to be a final concentration of 12.5 nanomolar (nM) 
Column 2 and Column 24 are neutral control columns (Contain: Enzyme, Substrate, DMSO; no experimental compounds)


SECTION: Prepare 384-well Plate
4.2. EMPTY Multi-Drop Combi Tube Dispensing Cassette (by selecting the EMPTY button on the Combi Dispenser until the tubes of the cassette are emptied). Discard the 50 nanomolar (nM) discharged from the cassette.
SECTION: Prepare 384-well Plate
6. CENTRIFUGE plate 1500 rpm, 1 min to remove bubbles
SECTION: Prepare 384-well Plate
7. INCUBATE plate for 15 min atRoom temperature 
SECTION: Prepare 384-well Plate
8. PRIME Multi-Drop Combi Tube Dispensing Cassette with1600 nanomolar (nM) by selecting the PRIME button on the Combi Dispenser until the tubes are filled completely. 
Note: Be sure to cycle dispensing several times on a clean plate lid (This confirms there are no bubbles in the Dispensing Cassette).
SECTION: Prepare 384-well Plate
8.1. DISPENSE 4 µL 1600 nanomolar (nM) into Columns 1-24 (full plate)

Note: 
 1600 nanomolar (nM) is four times the final concentration for the assay. It will be diluted to be a final concentration of400 nanomolar (nM) 


SECTION: Prepare 384-well Plate
8.2. EMPTY Multi-Drop Combi Tube Dispensing Cassette (by selecting the EMPTY button on the Combi Dispenser until the tubes of the cassette are emptied). Discard the 1600 nanomolar (nM) discharged from the cassette.
SECTION: Prepare 384-well Plate
9. CENTRIFUGE plate 1500 rpm, 1 min to remove bubbles
SECTION: Prepare 384-well Plate
10. PRIME Multi-Drop Combi Tube Dispensing Cassette with Assay Buffer by selecting the PRIME button on the Combi Dispenser until the tubes are filled completely. Then, EMPTY the Multi-Drop Combi Tube Dispensing Cassette (by selecting the EMPTY button on the Combi Dispenser until the tubes of the cassette are emptied). Discard the Assay Buffer discharged from the cassette.
SECTION: Prepare 384-well Plate
11. PRIME Multi-Drop Combi Tube Dispensing Cassette with 0.25 % volume  by selecting the PRIME button on the Combi Dispenser until the tubes are filled completely. 
Note: Be sure to cycle dispensing several times on a clean plate lid (This confirms there are no bubbles in the Dispensing Cassette).


SECTION: Prepare 384-well Plate
11.1. DISPENSE 8 µL 0.25 % volume into full plate

Note: 
 0.25 % volume is two times the final concentration for the assay. It will be diluted in the plate to be a final concentration of 0.125 % volume 
SECTION: Prepare 384-well Plate
11.2. EMPTY Multi-Drop Combi Tube Dispensing Cassette (by selecting the EMPTY button on the Combi Dispenser until the tubes of the cassette are emptied). Discard the 1600 nanomolar (nM) discharged from the cassette.
SECTION: Prepare 384-well Plate
12. CENTRIFUGE 1500 rpm, 1 min  plate to remove bubbles
SECTION: Prepare 384-well Plate
13. INCUBATE plate for 60 min at Room temperature 

Recommended: Clean/Empty the Multi-Drop Combi Reagent Dispenser and Dispensing Cassette during this incubation step
SECTION: Read Plate Fluorescence
14. READ and RECORD the plate Relative fluorescence units (RFU) via the "Mac1 Protocol" on the PHERAstar FS Control Software.
 

 

SECTION: Prepare 384-well Plate
5. PRIME Multi-Drop Combi Tube Dispensing Cassette with Assay Buffer by selecting the PRIME button on the Combi Dispenser until the tubes are filled completely. Then, EMPTY the Multi-Drop Combi Tube Dispensing Cassette (by selecting the EMPTY button on the Combi Dispenser until the tubes of the cassette are emptied). Discard the Assay Buffer discharged from the cassette.
SECTION: Diagram of assay
15. Figure 1 graphical depiction of assay principal and its use in screening campaign

 

SECTION: Experimental Design
16. 
 


SECTION: SARS-CoV-2 nsp3 expression and purification
1. The protein used in this assay was expressed and purified using the following protocol.