Apr 25, 2024

Public workspaceSARS-CoV-2 nsp3 macrodomain Time-Resolved FRET peptide displacement assay V.3

DOI
dx.doi.org/10.17504/protocols.io.eq2ly7r2mlx9/v3
SARS-CoV-2 nsp3 macrodomain Time-Resolved FRET peptide displacement assay
  • 1The Weizmann Institute of Science;
  • 2ASAP Discovery
  • Haim Barr: General acknowledgement: The Wohl Drug Discovery institute, The Nancy and Stephen Grand Israel National Center for Personalized Medicine.;
Mary-Ann Xavier
Open access
DOI: dx.doi.org/10.17504/protocols.io.eq2ly7r2mlx9/v3
Protocol Citation: Haim Barr, Noa Lahav 2024. SARS-CoV-2 nsp3 macrodomain Time-Resolved FRET peptide displacement assay. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly7r2mlx9/v3Version created by Mary-Ann Xavier
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 05, 2024
Last Modified: April 25, 2024
Protocol Integer ID: 97835
Keywords: Coronaviridae, Time-Resolved FRET assay, Fluorescence assay
Funders Acknowledgement:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
Disclaimer
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.


Abstract
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 is intended to measure the activity of Mac1 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 and inhibitors which displace the peptide will prevent energy transfer. Binding activity is reported as the ratio of Acceptor/Donor (Em/Em) X 10,000.
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Experiment Concentrations (From Stock to Assay)
ABCDE
ReagentStockLoaded into CombiFinal in assay plateUnits
His-SARS CoV-2 MAC1183000 5012.5nM
Substrate (Biotin-ADPr) 100000001600400nM
Detection solution
Streptavidin-XL665 (SA-XL)10.250.125%
MAb Anti-6HIS-Eu cryptate Gold1000.250.125%
Assay buffer
HEPES pH=7.025025 25 mM
NaCl200 20 20 mM
BSA0.50.050.05mg/ml
Tween 20 0.50.050.05%
HTRF PPI Europium Detection Buffer1001010 %
For more information, please check out the "Materials" Section
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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
Materials
Assay Buffer Reagents (Concentration listed are from Stock Solutions)
  1. Concentration250 millimolar (mM) ReagentHEPES 0.5M buffer soln. pH 7.0Fisher ScientificCatalog #AAJ60064AE (or similar)
  2. Concentration200 millimolar (mM) ReagentSodium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9888 (or similar)
  3. Concentration0.5 mg/mL ReagentBovine Serum Albumin (BSA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A7030
  4. Concentration0.5 % volume ReagentTWEEN® 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P9416
  5. Concentration100 % volume ReagentHTRF PPI Europium Detection BufferCISBIO BIOASSAYS, company of PerkinElmerCatalog #61DB9RDF
*Note: There are several forms of the Assay Buffer in this experiment. The Assay Buffer is the final, active buffer used throughout the experiment and has all of the five above reagents included. HTRF PPI Europium Detection Buffer needs to be added fresh before each experiment. Thus, there was an intermediate Buffer called Mac1 Buffer that contained HEPES, NaCl, BSA, and Tween only. Mac1 Buffer was filtered and stored at 4˚C. HTRF PPI buffer was then added to Mac1 Buffer fresh (to a final concentration of 10%) prior to performing the experiment--creating the active Assay Buffer.
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Detection Solution Reagents (Concentration listed are from Stock Solutions)
Concentration1 % volume ReagentStreptavidin-XL665CISBIO BIOASSAYS, company of PerkinElmerCatalog #610SAXAC
  • Note: Streptavidin-XL665 was dissolved in triply distilled water and diluted with HTRF PPI buffer to its stock concentration and then was aliquoted into 1.5mL sterile conical tubes
Concentration100 Mass Percent ReagentMAb Anti-6HIS-Eu cryptate GoldCISBIO BIOASSAYS, company of PerkinElmerCatalog #61HI2KLA
Note: MAb Anti-6HIS-Eu cryptate Gold was dissolved in tripled distilled water and then aliquoted into 1.5mL sterile conical tubes

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Additional Reagents:
Concentration183000 nanomolar (nM) His-SARS COV2 MAC1 Enzyme
  • The Enzyme stock was originally Concentration183000 nanomolar (nM) and was diluted to Concentration50 nanomolar (nM) before every experiment in freshly made Assay Buffer. The final assay concentration is Concentration12.5 nanomolar (nM)
Concentration10000000 nanomolar (nM) Substrate (Biotin-ADPr) MAC1
  • Substrate stock (ARTK(Bio)QTARK(Aoa-RADP)S) was dissolved in DMSO to the stock concentration. Before each experiment, the substrate stock was diluted to Concentration1600 nanomolar (nM) in freshly made Assay Buffer.

Safety warnings
Attention
Please be sure to wear proper Personal Protective Equipment (PPE) while performing this experiment.
Before start
Note: Inhibitor compounds stock concentration is Concentration20 millimolar (mM) . Compounds are pre-dispensed into 384 plates and stored at -20˚C until use.
Prepare Reagents
Prepare Reagents
PREPARE all of the reagents/buffers required for this experiment.

Assay Buffer
ABCDE
ReagentStockLoaded into CombiFinal in assay plateUnits
HEPES pH=7.025025 25 mM
NaCl200 20 20 mM
BSA0.50.050.05mg/ml
Tween 20 0.50.050.05%
HTRF PPI Europium detection buffer1001010%
Reagents (dilute reagents in assay buffer for required volume)
ABCDE
ReagentStockLoaded into CombiFinal in assay plateUnits
His-SARS-CoV-2 MAC1183000 5012.5nM
Substrate (Biotin-ADPr) 100000001600400nM
Detection Solution (dilute reagents in assay buffer for required volume)
ABCDE
ReagentStockLoaded into CombiFinal in assay plateUnits
Streptavidin-XL665 (SA-XL)1 0.250.125%
MAb Anti-6HIS-Eu cryptate Gold1000.250.125%

Prepare 384-well Plate
Prepare 384-well Plate
16m
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 a clean plate lid (This confirms there are no bubbles in the Dispensing Cassette).
DISPENSE Amount4 µL Mac1 Buffer to Columns 1 and 23 of assay plate
  • Note: These will represent the inhibitor control columns

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.
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 a clean plate lid (This confirms there are no bubbles in the Dispensing Cassette).
DISPENSE Amount4 µL Concentration50 nanomolar (nM) His-SARS COV2 MAC1 Enzyme to Columns 2-22 and 24 of assay plate
Note:
  • Concentration50 nanomolar (nM) His-SARS COV2 MAC1 is four times the final concentration for the assay. It will be diluted to be a final concentration of Concentration12.5 nanomolar (nM) His-SARS COV2 MAC1 Enzyme
  • Column 2 and Column 24 are neutral control columns (Contain: Enzyme, Substrate, DMSO; no experimental compounds)


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 Concentration50 nanomolar (nM) His-SARS COV2 MAC1 Enzyme discharged from the cassette.
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.
CENTRIFUGE plate Centrifigation1500 rpm, Room temperature, 00:01:00 to remove bubbles
1m
INCUBATE plate for Duration00:15:00 atTemperatureRoom temperature
15m
PRIME Multi-Drop Combi Tube Dispensing Cassette withConcentration1600 nanomolar (nM) MAC1 Substrate (Biotin-ADPr) 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 a clean plate lid (This confirms there are no bubbles in the Dispensing Cassette).
DISPENSE Amount4 µL Concentration1600 nanomolar (nM) MAC1 Substrate (Biotin-ADPr) into Columns 1-24 (full plate)

Note:
  • Concentration1600 nanomolar (nM) MAC1 Substrate (Biotin-ADPr) is four times the final concentration for the assay. It will be diluted to be a final concentration ofConcentration400 nanomolar (nM) MAC1 Substrate (Biotin-ADPr)


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 Concentration1600 nanomolar (nM) MAC1 Substrate (Biotin-ADPr) discharged from the cassette.
CENTRIFUGE plate Centrifigation1500 rpm, Room temperature, 00:01:00 to remove bubbles
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.
PRIME Multi-Drop Combi Tube Dispensing Cassette with Concentration0.25 % volume Detection Solution 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 a clean plate lid (This confirms there are no bubbles in the Dispensing Cassette).


DISPENSE Amount8 µL Concentration0.25 % volume Detection Solution into full plate

Note:
  • Concentration0.25 % volume Detection Solution is two times the final concentration for the assay. It will be diluted to be a final concentration of Concentration0.125 % volume Detection Solution
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 Concentration1600 nanomolar (nM) MAC1 Substrate (Biotin-ADPr) discharged from the cassette.
CENTRIFUGE Centrifigation1500 rpm, Room temperature, 00:01:00 plate to remove bubbles
INCUBATE plate for Duration01:00:00 at TemperatureRoom temperature

Recommended: Clean/Empty the Multi-Drop Combi Reagent Dispenser and Dispensing Cassette during this incubation step
1h
Read Plate Fluorescence
Read Plate Fluorescence
READ and RECORD the plate Relative fluorescence units (RFU) via the "Mac1 Protocol" on the PHERAstar FS Control Software.
Equipment
PHERAstar FS
NAME
Microplate reader
TYPE
BMG LABTECH
BRAND
0471B0001A
SKU
https://www.bmglabtech.com/en/pherastar-fsx/?utm_term=pherastar%20plate%20reader&utm_campaign=usa.roi.products&utm_source=adwords&utm_medium&gclid=Cj0KCQjw8qmhBhClARIsANAtbodGRjigZtEYwcoMXUtxsLn25xp4gjKra3ZNt9jLh9-FwOoFR_5EUHUaAlkREALw_wcB
LINK

Expected result
Donor 325/620 ex/em should be ~ 5000 . Acceptor ~ 3000

Diagram of assay
Diagram of assay
Figure 1 graphical depiction of assay principal and its use in screening campaign

Principal of ADPr peptide displacement assay A - Binding of ADPr-modified peptide to Mac1 protein B- Detection reagents added to protein+peptide complex C- TR-FRET detects binding of peptide to Mac1 if proximity of donor and acceptor detection reagents is sufficient to enable resonant energy transfer D- Inhibitor compounds are detected by reduced TR-FRET signal when inhibitor displaces/prevents binding of ADPr-peptide to Mac1
Principal of ADPr peptide displacement assay
A - Binding of ADPr-modified peptide to Mac1 protein B- Detection reagents added to protein+peptide complex C- TR-FRET detects binding of peptide to Mac1 if proximity of donor and acceptor detection reagents is sufficient to enable resonant energy transfer D- Inhibitor compounds are detected by reduced TR-FRET signal when inhibitor displaces/prevents binding of ADPr-peptide to Mac1

Experimental Design
Experimental Design

384 plate layout
384 plate layout


Keywords
Keywords
Mac1, Nsp3, TR-FRET, HTRF, ADPr, Displacement, Screening, Assay, Inhibitor, Fragment, Binding, Macrodomain
Protocol references