Addressing Protein-Induced Fluorescence Enhancement in Cy3-Cy5 FRET Studies: Improving Distance Measurements in Protein-DNA Complexes

The cyanine fluorophore pair Cy3 and Cy5 is widely used in biophysical research to measure molecular distances via Förster Resonance Energy Transfer (FRET). However, Protein-Induced Fluorescence Enhancement (PIFE) can significantly affect the accuracy of FRET measurements involving these dyes. In this study, we investigate how PIFE impacts the determination of DNA-DNA distances within the Sleeping Beauty transposase-DNA complex. By analyzing FRET efficiency changes, we demonstrate that PIFE leads to inaccuracies in experimentally measured distances. To address this issue, we propose two mitigation strategies: (1) Altering labeling sites to positions less susceptible to PIFE, and (2) Employing PIFE-insensitive fluorophores like BODIPY-TMR as the donor dye when labeling site substitution is impractical. Our results show that both approaches effectively reduce PIFE's influence, leading to more accurate distance measurements. This work underscores the importance of accounting for PIFE in FRET studies and provides practical solutions to enhance the reliability of molecular distance determinations in protein-DNA complexes.