Molecular Dynamics Study of FRET Orientation Factors to Infer the Structural Deformations in Damaged DNA

Abstract In Förster resonance energy transfer (FRET) experiments for probing DNA structural deformations, the orientation factor κ2 is critical for accurate distance estimation. Although κ2 depends on the molecular behavior of fluorescent dyes, its quantitative variation remains insufficiently understood. To elucidate this effect at the molecular level, we performed molecular dynamics (MD) simulations of gap-containing DNA with Alexa Fluor 488 and 647 dyes attached to the DNA termini via linker fragments. An undamaged DNA system was also simulated using the Cy3/Cy5 dye pair. For each MD snapshot, dye configurations were classified into adsorbed and desorbed states based on the solvent-accessible surface area. This analysis revealed distinct κ2 behaviors upon dye adsorption and desorption; in the desorbed state, <κ2> was close to the isotropic limit of 2/3, whereas adsorption led to a significant reduction (<κ2> ≈ 0.46–0.58). These results indicate that MD-derived estimates of <κ2> are preferable for accurate distance estimates. In contrast, the commonly used isotropic approximation <κ2> = 2/3 may introduce distance errors of ∼5 Å; however, such errors are relatively modest and may be acceptable depending on the required accuracy. In addition, the present MD simulations revealed DNA conformational deformations induced by gap lesions and their effects on <κ2>.