Dear colleagues,
I am working on SARS-CoV-2 Mpro (3CL protease). I am currently using a peptide substrate that has FAM as a fluorophore and Dabcyl as a quencher. The system starts to fluoresce once the substrate is cut by Mpro. However, I would like to invert the assay so that the substrate fluoresces only when intact. Could anyone perhaps recommend a suitable FRET pair based on the peptide length, etc.?
Currently used substrate: https://www.discoverypeptides.com/5-fam-lys-dabcyl-cov-main-protease-mpro-substrate
Mpro: https://www.sigmaaldrich.com/LT/en/product/sigma/sae0172
To make that work, you would have to monitor the fluorescence of the acceptor rather than the donor. This method typically results in a smaller signal than monitoring the increase in donor fluorescence. Replace the Dabcyl with TAMRA. When the substrate is cut, the FAM fluorescence will decrease and the TAMRA fluorescence will increase (slightly) when exciting FAM. If you measure the ratio of TAMRA to FAM fluorescence, you will get a large improvement in signal-to-noise ratio compared with measuring a single intensity. However, this comes at the cost of a nonlinear relationship of product formation to signal. For a full explanation, please see this paper:
Article A homogeneous, high-throughput fluorescence resonance energy...
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