The bottom line:
We have found a way to link biotin to a variety of compounds using a bond that is resistant to enzyme degradation, while maintaining biotin's ultra-high affinity to streptavidin.
So why should you care? Biotin is commonly used in biology labs due to its incredibly strong and specific binding to a glycoprotein called streptavidin. Due to this, you can tag a protein/compound with biotin then use streptavidin “hooks” to fish it out of otherwise complex mixtures.
Now, this works fine in the lab but in bodily fluids, an enzyme called biotinidase shows up and ruins the party by quickly breaking the bond between the compounds and the biotin attached to them. Using our link, we prevent this, while maintaining the strong affinity between the biotinylated compound and its hook (which is usually reduced with existing degradation-resistant linkers
). This makes it possible to explore applications where biotinylated compounds are exposed to biological fluids (especially blood) for long periods of time (and some other cool stuff – see below).
We’d love to hear what you could come up with in terms of specific applications in the fields below or anywhere else. Given how widely used biotin is in biology, there must be a myriad of other applications out there we haven’t considered yet – can’t wait to hear what you come up with!
A great idea would:
- Make use of the linker’s unique advantage, i.e. that it retains high specificity while being resistant to biotinidase;
- Have strong commercial potential (what’s the market?);
- Include some specifics on how to move the idea forward, i.e. technical details, potential industry partners etc.
The main applications we have considered revolve around so-called “pre-targeting” which results in a greater ratio of the compound in the target tissue vs. the bloodstream, in e.g. imaging and radiotherapy.
There are several other applications where the increased enzyme-resistivity without loss of high affinity for streptavidin can be useful:
- Biotinylation of peptides/proteins for array display
- Affinity-purification mass spectrometry with increased ultimate purity.
- More accurate fluorescent biotinidase assay (to detect e.g. biotinidase deficiency in newborns)
You can find more information as well as some references on each of these applications in the exandable sections below.
About the linker
We have developed a way to bind biotin to a variety of compounds via triazole bonds, which are highly resistant of enzymes, which is relevant in a number of settings as discussed above. Using click chemistry means biotin is attached in a highly efficient and selective manner, with no interference from/with other biological compounds. Furthermore, the triazole bond may be combined with a range of linkers between the bond and the compound, for example PEG to increase water solubility, light- or enzyme-cleavable linkers, etc.
The main applications we have considered revolve around pre-targeting: Here a biotinylated antibody is allowed to bind to a site of interest (e.g. a tumour), followed by addition of streptavidin, which binds to the exposed biotin. Then the biotinylated compound of interest (e.g. an imaging label or radiotherapeutic) is injected and allowed to bind to this streptavidin in turn. These smaller compounds of interest are vulnerable to degradation by biotinidase – so this is where our new coupling technique is important. This pre-targeting approach results in a greater ratio of the compound in the target tissue vs. the bloodstream, which is useful in both e.g. imaging and radiotherapy.
About the other potential applications
- Biotinylation of peptides/proteins for array display: The functionalized proteins/peptides can be captured by a streptavidin-functionalized substrate efficiently and retain their activity.
- Affinity-purification MS: biotinylated peptides resulting from a trypsin digest are captured by a streptavidin-functionalised matrix and subsequently identified using mass spectrometry. With a degradation-resistant linkage we can introduce a purification step to remove any endogenously biotinylated peptides, increasing ultimate purity.
- Fluorescent biotinidase assay (to detect e.g. biotinidase deficiency in newborns): Combine biotinidase-resitant and –susceptible fluorophores in an accurate ratiometric assay.