Figure 1.

Basic principles of cpFP-based biosensors. (A) The scheme of FP circular permutation at the DNA and protein levels. The resulting cpFP can be integrated between the interacting units (1 and 2) of the sensory domain. Units 1 and 2 can be different proteins, subunits or domains of the same protein. (B–D) Variants of cpFP-based biosensors with ratiometric readout. (B) The ratiometric biosensor can be developed on the basis of a single cpFP, since some are characterized by the presence of two peaks in the fluorescence excitation spectrum. (C) A second FP with different spectral properties can be attached to the N- or C-termini of a biosensor. (D) (Green-Orange)-Matryoshka cassette for designing ratiometric biosensors.