New specific fluorescent labelling method

The technology consists of novel compounds containing two maleimide groups attached via a tether to a fluorescent core, whose latent fluorescence is quenched until their maleimide groups undergo a specific thiol addition reaction. Complementary proteins are designed bearing two cysteine residues appropriately positioned to react with our novel fluorogens.

Maleimide groups are known to react selectively with thiols via addition reactions involving their C=C double bond. They are also known to quench fluorescence in their conjugated form, but not as their thiol adduct products. Thus, the fluorogens were prepared bearing two maleimide groups, so their latent fluorescence would only be realized upon their reaction with two thiol equivalents. Furthermore, the positioning of maleimide groups is such that they are separated by a precise distance. The resulting fluorogen reacts rapidly and specifically with compounds presenting two sulfhydryl groups separated by the appropriate distance.

The technology allows for protein localization and protein-protein interaction studies in living cells. More specifically, this technique provides detection of the following:

• proteins in appropriate sub-cellular compartments or organelles (localisation, turnover, trafficking)
• protein-protein interactions in vivo and in vitro in any cell type
• proteins in physiological cellular conditions detectable in real-time
• proteins in-gel, eliminating protein staining and western blot procedure.

Currently, 27 fluorogens are available. But, virtually any fluorophore could be attached to dimaleimide moieties of different dimensions.

Competitive advantages
This technology possesses several advantages:
• High specificity and sensitivy: maleimide groups react selectively with thiols
• Safer – maleimide groups are less toxic than the organoarsenic-based fluorogens prevalently used
• No false positive results – any background reaction with proteins bearing only one cysteine residue would not lead to increased fluorescence since the monothiolated fluorogen does not fluoresce
• Small tag (~2 kD) – unlikely to disrupt protein function or protein-protein interaction
• Multiplex labelling – different fluorogen/target sequence pairs (based on varied distances between the maleimide groups and Cys residues) allows simultaneous labelling of different proteins with different colours
• Allows intracellular labelling due to the permeability of our synthetic fluorogens