REAGENTLESS FLUORESCENT BIOSENSORS COMPRISING A DESIGNED ANKYRIN REPEAT PROTEIN MODULE, RATIONAL DESIGN METHODS TO CREATE REAGENTLESS FLUORESCENT BIOSENSORS AND METHODS OF THEIR USE
The present invention relates to reagentless fluorescent biosensors which comprise at least one ankyrin repeat and a fluorophore and are specific for at least one target; the method for preparing such reagentless fluorescent biosensors comprises the following steps: (a) identifying the residues (R1) of the paratope of the biosensor by mutagenesis of all, or of a subset, of the residues of the biosensor, and determining variations in at least one measurable chemical or physical parameter of interaction with said at least one target; wherein said variations are due to each mutation or to groups of mutations; (b) selecting the cysteine residues, or the residues to be mutated to cysteine, from the residues (R2) of the biosensor which are located adjacent to the. residues of the paratope; (c) mutating by site-directed mutagenesis at least one of the residues (R2) selected in (b) to a cysteine residue when said residue is not naturally a cysteine residue; and (d) coupling the S? atom of at least one cysteine residue (R2) obtained in (b) or in (c) to a fluorophore.