Development of FRET-based RCC1 biosensor. (A) Schematic of the FRET-based RCC1 biosensor CFP-RCC1-YFP. (B) The biosensor CFP-RCC1-YFP exhibits efficient FRET. Excitation was at 430 nm. Emission spectra are shown for CFP alone, YFP alone, and for CFP-RCC1-YFP. (C) FRET efficiency of CFP-RCC1-YFP is saturably reduced by Impα3; representative emission spectra ± Impα3. (D) Titration of the emission ratio (YFP/CFP) versus increasing concentrations of Impα3. (E) DNA decreases FRET efficiency of CFP-RCC1-YFP in a dose-dependent manner. Annealed 64-nt complimentary oligonucleotides were used as the source of DNA. FRET efficiencies were measured in the presence of increasing concentrations of oligonucleotides. Emission spectrum is shown ± DNA. (F) Titration of emission ratio (YFP/CFP) versus increasing DNA concentration. For D and F, error bars represent ±1 SD.