Team:IISER Mohali/Choice Of Fluorophores
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Choice of Fluorophores
Fluorophore Mechanism:
ENGINEERING FLUOROPHORE INSERTION-
The designed Encapsulin is adhered to the testing vial surface and has a substrate on the outside designed to be more preferably recognized and cleaved by MMP9 and will be loaded with a ligand-switchable fluorophore loaded from the inside by targetting peptide.
- PRINCIPLE-
The size of the ligand which can activate the fluorophore is such that it cannot enter the normal pores of Encapsulin nanocage (largest pore size for the Encapsulin of our choice is around 1 nm, hence, the ligand is at least or more than 1 nm), hence no fluorescence will be detected
But, when there is MMP9 present in the surrounding mobile medium, then it will dock to the substrate and cleave it, creating a pore size enough for the ligand to enter and activate the fluorophore, thereby getting the fluorescence signal.
However, it had a major problem that was yet not resolved, which was about the fluorophore that can be taken as a candidate for this set-up.
- CONSTRAINTS-
- The candidate fluorophore should be switchable with a ligand that is slightly greater than 1 nm, but should not be too large (say, > 5nm).
- Appropriate fluorophore properties such as brightness index and maximum absorbance wavelength (choice and price of laser),
- Size of the fluorophore.
- PROSPECTIVE CANDIDATES-
After the selection of our project idea, we geared up to find the potential candidate fluorophore. On doing the literature survey, survey, we considered two fluorophores: 4-CN-Trp (4-Cyano-Tryptophan) and 5-CN-Trp.
However these are not naturally occurring amino acids, hence, production or incorporation might become costly, and they do not really have that large of differential fluorescence intensity as may be required in a diagnostic kit.
Another candidate was UnaG. UnaG shows the switchability in fluorescence by a small-molecule, bilirubin, which is supposed of size >= 1 nm, hence making it promising, although skeptically. However, upon further inspection, UnaG has a fatal flaw that could be unwanted for our use, which is the ability to degrade itself and protein of interest tagged with it, if bilirubin is not bound to it.
Finally, after going through more literature, one of us got inspiration from fluorescent fusion proteins and iso-peptide covalent bond formation.
- PREFERRED MECHANISM-
Rather than have a switchable fluorophore, we reasoned that we can use any fluorophore of appropriate size, and add a peptide tag that has the potential to bind to another protein domain (known in literature). This domain can actually be loaded inside the Encapsulin using TP (targetting peptide).
Hence, once the substrate is cleaved, the fluorophore can enter, and from the peptide tag, it will adhere to the protein domain loaded. After a washing step, excess unbound fluorophore will be removed and the fluorophore that still remains are those which have bound with protein domain subject to entry inside the nanocage which depends on how many nanocages were cleaved, which itself depends on what the concentration of MMP9 is.
Thus, one can estimate the levels of MMP9. One more benefit that this set-up has is in terms of modularity of parts which is one of the important requirements of synthetic biology parts.
The one that was finalized was the SpyTag003 peptide tag and SpyCatcher003 protein domain after comparative analyses of various candidates.
SELECTING THE FLUOROPHORES-
Since we also had to choose a fluorophore to use with it, we referred to Fpbase and screened out fluorophores that had -
- Brightness index >= 70 units
- Maximum excitation wavelength >= 500 nm
- Oligomerization state being monomeric.
Upon the search, we essentially found 7 candidates. The entire list of fluorophores shortlisted can be found here
After the list was created, we began exploring each one of them one-by-one properly, so that we can find which fluorophore to go with, after a comparative analysis of all these candidates.
Finally, due to mNeonGreen having almost all of the favorable characteristics and also being highly characterized, we decided to work with it for our kit.
However, we also noted that since this kit is modular in this aspect, we could easily replace any other fluorophore which might be better and is of appropriate size.
REFERENCES
- Blue fluorescent amino acid. Mary Rose Hilaire, Ismail A. Ahmed, Chun-Wei Lin, Hyunil Jo, William F. DeGrado, Feng Gai. Proceedings of the National Academy of Sciences Jun 2017, 114 (23) 6005-6009; DOI: 10.1073/pnas.1705586114
- https://www.microscopyu.com/tutorials/blue-excitation
- https://www.fpbase.org/
- https://www.ebi.ac.uk/merops/