Jun 20, 2025
Version 1
Enteroviruses (EV-D68/EV-A71) 3C and EVA71-2A proteases fluorescence single and dose response V.1
- Haim Barr1,2,
- Noa Lahav1,2
- 1Weizmann Institute of Science;
- 2ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Haim Barr, Noa Lahav 2025. Enteroviruses (EV-D68/EV-A71) 3C and EVA71-2A proteases fluorescence single and dose response. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8kb5l5r/v1
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: May 01, 2024
Last Modified: June 20, 2025
Protocol Integer ID: 99073
Keywords: EV-D68, EV-A71, 3C protease, Enterovirus, biochemical assay, functional assay, infectious disease, 2A protease, enteroviruses infection, enterovirus, compounds top assay concentration, compounds assay concentration, treatments for viral infectious disease, biochemical assay, top assay concentration, assay protocol, a71 2a protease, viral infectious disease, direct enzyme activity measurement method, enzyme activity, assay reliability, point assay, assay, total assay volume, fluorescence intensity, dose response point, dose response for ic50 measurement, compound plate design for dose response, fluorescence, 2a protease
Funders Acknowledgements:
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 is a functional, biochemical assay used to identify treatments for viral infectious diseases related to Enteroviruses infection, specifically EV-D68 3C, EV-A71 3C and EV-A71 2A proteases. In this current version we have added the Addgene id.
Utilizing a direct enzyme activity measurement method, the experiment was performed in a 384-well plate reading the fluorescence intensity. This assay tested the mode of action of inhibition.
Initial screening was conducted with 2 compound concentrations to determine %inhibition, followed by dose response for IC50 measurement (as specified below). In each run a reference compound was added to verify the assay reliability and robustness.
Assay protocol was developed at the Weizmann Institute of Science, as a part of the ASAP Discovery Consortium. In this curre
Compounds Plate Design for 2-Point Assay:
Total Assay Volume: 20 µL
Compounds Assay Concentration: 100 µM and 50 µM
Dilution Factor: 2
Dose Response Points: 2
Number of Replicates: 2
Backfill with DMSO: Yes
Compound Plate Design for Dose Response:
Total assay volume: 20 µL
Compounds top assay concentration: 100 µM
Dilution factor: 2
Dose response points: 12
Number of replicates: 2
Backfill with DMSO: yes
Materials
Assay Buffer Reagents (Concentration listed are Stock Solution Concentrations)
- 50 Mass / % volume Glycerol - for molecular biology, ≥99%Merck MilliporeSigma (Sigma-Aldrich)Catalog #G5516
- 1000 millimolar (mM) Tris(2-carboxyethyl)phosphine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #75259
- 100 millimolar (mM) Sodium ChlorideFisher ScientificCatalog #S271 (or similar)
- 10 mg/mL BSA-Molecular Biology Grade - 12 mgNew England BiolabsCatalog #B9000S (or similar)
- 1000 millimolar (mM) TCEP HClP212121Catalog #SV-TCEP (or similar)
- 10 % volume Tween 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P1379 (or similar)
*Note: all components are added fresh to the assay buffer before each experiment
---------------------------------------------------------
Additional Reagents:
Proteases and substrate:
- 3C Protease stock concentration was 1340 micromolar (µM) for EV-D68 and 1400 micromolar (µM) for EV-A71 3C. Stocks were diluted to their assay concentrations with freshly made Assay Buffer before each experiment
- The substrate for EV-D68/EV-A71 3C proteases was 5FAM- KEALFQGPPQFE-DABCYL (custom made by GenScript Biotech (Singapore) PTE. LTD).
- The substrate for EV-A71 2A protease was Dabcyl-KITTLGKFGQDE-Edans (custom made by Peptide 2.0 Inc.).
- Substrate stock was created by dissolving the substrate powder in DMSO to create 10 millimolar (mM) Substrate Stock aliquots and stored at -80 °C . Before each experiment, the Substrate Stock was diluted to its assay concentration with freshly made Assay Buffer.
Protocol materials
Glycerol - for molecular biology, ≥99%Merck MilliporeSigma (Sigma-Aldrich)Catalog #G5516
Sodium ChlorideFisher ScientificCatalog #S271
BSA-Molecular Biology Grade - 12 mgNew England BiolabsCatalog #B9000S
Tween 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P1379
Tris(2-carboxyethyl)phosphine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #75259
TCEP HClP212121Catalog #SV-TCEP
EV-A71 3C protease; aliases: Enterovirus A71 3C protease, non cleavable C-term 6xHis tagaddgeneCatalog #204816
EV-D68 3C protease; aliases: Enterovirus A71 3C protease, non cleavable C-term 6xHis tagaddgeneCatalog #(Plasmid #204817)
Enterovirus Coxsackievirus A16 2A proteaseaddgeneCatalog #228632
Enterovirus A71 3C proteaseaddgeneCatalog #204816
EV-D68 3C protease; aliases: Enterovirus A71 3C protease, non cleavable C-term 6xHis tagaddgeneCatalog #204817
Human Coxsackievirus A16 strain G10 2A proteaseaddgeneCatalog #228632
Safety warnings
Please be sure to wear proper Personal Protective Equipment (PPE) while performing this experiment.
Before start
Dummy plate: In each step of the protocol, dispense the reagents into an empty plate before dispensing them into the assay compounds plate (this will detect and prevent dispensing issues).
Note: Inhibitor compounds stock concentration is 20 mM. Compounds are pre-dispensed into 384 plates and stored at -20˚C until use.
Protein expression and purification
Expression and purification protocol and the plasmid used for EV-A71 3C proteaseEnterovirus A71 3C proteaseaddgeneCatalog #204816
Protocol
CREATED BY
korvus.wang
Expression and purification protocol for the plasmid used EV-D68 3C protease EV-D68 3C protease; aliases: Enterovirus A71 3C protease, non cleavable C-term 6xHis tagaddgeneCatalog #204817
Protocol
CREATED BY
korvus.wang
Expression and purification for the plasmid used EV-A71 2A protease Human Coxsackievirus A16 strain G10 2A proteaseaddgeneCatalog #228632
Protocol
CREATED BY
korvus.wang
Prepare Reagents
PREPARE all of the reagents/buffers required for this experiment.
| A | B | C | D | E | |
| Reagent | Stock Concentration | Concentration Loaded into dispenser | Final Concentration in plate | Units | |
| EV-D68 3C Enzyme stock | 1340 | 0.04 | 0.02 | uM | |
| EV-A71 3C Enzyme stock | 1400 | 0.3 | 0.15 | uM | |
| EV-A71 2A Enzyme stock | 1200 | 0.4 | 0.2 | uM | |
| EV-D68 3C Substrate | |||||
| EV-A71 3C Substrate | 10000 | 9 | 4.5 | uM | |
| EV-A71 2A Substrate | 10000 | 12 | 6 | uM | |
| Assay Buffer | |||||
| Tris (pH 7.5) | 250 | 50 | 50 | mM | |
| Sodium Chloride | 5000 | 150 | 150 | mM | |
| BSA | 10 | 0.05 | 0.05 | mg/mL | |
| Tween 20 | 10 | 0.1 | 0.1 | % by volume | |
| TCEP | 1000 | 0.5 | 0.5 | mM | |
| Glycerol | 50 | 10 | 10 | % by volume | |
For more information, please see the Materials sec
We used the following plasmid in the protein expression and purification protocol.
EV-A71 3C protease; aliases: Enterovirus A71 3C protease, non cleavable C-term 6xHis tagaddgeneCatalog #204816
EV-D68 3C protease; aliases: Enterovirus A71 3C protease, non cleavable C-term 6xHis tagaddgeneCatalog #(Plasmid #204817)
Enterovirus Coxsackievirus A16 2A proteaseaddgeneCatalog #228632
Prepare 384 Well Plate
32m
PRIME the dispenser with Assay Buffer by selecting the PRIME button until the tubes are filled completely.
DISPENSE 10 µL Assay Buffer to Columns 1 and 23 of assay plate
Note: These will represent the inhibitor control columns (Contain: Substrate + Assay Buffer + DMSO, no enzyme, no compounds)
EMPTY the dispenser tubes.
- Discard Assay Buffer discharged from the tubes.
PRIME the dispenser with 40 nanomolar (nM) EV-D68 Enzyme or 300 nanomolar (nM) EV-A71 Enzyme by selecting the PRIME button until the tubes are filled completely.
- Note: Be sure to cycle dispensing on an empty plate first to detect and avoid dispensing issues.
DISPENSE 10 µL 40 nanomolar (nM) EV-D68 Enzyme , 300 nanomolar (nM) EV-A71 3C Enzyme or 400 nanomolar (nM) EV-A71 2A Enzyme
to Columns 2 - 22 and Column 24
Note:
- Enzyme concentrations dispensed to the plate are two times the final concentration in the plate for the assay.
- Plate final concentrations are 20 nanomolar (nM) D68 Enzyme ,150 nanomolar (nM) EV-A71 Enzyme and 400 nanomolar (nM) EV-A71 2A Enzyme .
- Column 2 and Column 24 are neutral control columns (Contain: Enzyme, Substrate, DMSO, no compounds)
EMPTY the dispenser tubes.
- Discard the liquid discharged from the tubes.
CENTRIFUGE plate 1500 rpm, Room temperature, 00:01:00 to remove bubbles
1m
INCUBATE plate for 00:30:00 at Room temperature
⚠ Make sure the plate is protected from light!
During Incubation: wash and prepare the dispenser to the next step
30m
PRIME the dispenser with 9 micromolar (µM) EV-A71 or 6 micromolar (µM) EV-D68 Substrate by selecting the PRIME button until the tubes are filled completely.
- Note: Be sure to cycle dispensing on an empty plate to detect and avoid dispensing issues.
DISPENSE 10 µL EV Substrate into Columns 1 - 24 (the full plate)
Note:
- Be sure to cycle dispensing on an empty plate first to detect and avoid dispensing issues.
- EV Substrate concentration is two times the final assay concentration and diluted x2 with the enzyme/buffer in the plate.
CENTRIFUGE plate 1500 rpm, Room temperature, 00:01:00 in plate centrifuge to remove bubbles
1m
INCUBATE plate at Room temperature for 1 hr for EV-A71 3C, 30 min for EV-D68 and 45 min for EVA71-2A enzyme .
⚠ Make sure the plate is protected from light!
Recommended: Clean the dispenser during this incubation step
Read Plate Fluorescence
READ and RECORD the plate Relative fluorescence units (RFU) on the PHERAstar FS Control Software.
- Software is a standard Fluorescence Assay set for Optimal excitation wavelength 485 nm, emission wavelength 528 nm for 3C proteases and 360/470 for EVA71 2A protease.
Equipment
PHERAstar FS
NAME
Microplate reader
TYPE
BMG LABTECH
BRAND
0471B0001A
SKU
LINK
Expected result
For EV-A71: Gain 50 should yield ~32,000 RFU in full reaction and ~15,000 RFU in Buffer Control
For EV-D68: Gain 50 should yield ~37,000 RFU in full reaction and ~10,000 RFU in Buffer Control
Protein expression and purification
2d
For the EV-D68 3C protease we used the following protocol
Protocol
CREATED BY
korvus.wang
2d
Protocol references
Tan, Jinzhi et al. “3C protease of enterovirus 68: structure-based design of Michael acceptor inhibitors and their broad-spectrum antiviral effects against picornaviruses.” Journal of virology vol. 87,8 (2013): 4339-51. doi:10.1128/JVI.01123-12