Reagentless fluorescent biosensors from nanofitins, rational design methods to create reagentless fluorescent biosensors and methods of their use

摘要

The present invention relates to reagentless fluorescent biosensors from Nanofitins which comprise at least an OB-fold protein and a fluorophore and are specific for a target ; the method for preparing such reagentless fluorescent biosensors comprises: (a) substituting each of the variable residues of said OB-fold protein, individually with a cysteine residue, thereby obtaining a set of cysteine mutants; (b) coupling said cysteine residue of each of the cysteine mutants of step (a) to a fluorophore, thereby obtaining biosensors; (c) purifying the biosensors of step (b); (d) measuring the equilibrium constant (K_D) between each of said purified biosensors of step (c) and said target, or the dissociation (K_off) and association (k_on) rate constants for each of said biosensors of step (c) and said target; and measuring the fluorescence variation of each of said biosensors of step (c) between a free and target bound state; and (f) determining the sensitivity (s) and/or relative sensitivity (s_r) of each of said biosensors of step (c) from the measurements of step (d).