Fig. 8. Distance and orientation dependence of FRET. (A) Time-resolved fluorescence anisotropy of free Cy3 (blue: fast rotation), Cy3 attached to dsDNA through a C6 flexible linker (red: fast and slow rotation), and DNA backbone internal modification (black: slow rotation). Reproduced with permission from ref. 154 Copyright 2009 American Chemical Society. (B) EFRET for Cy3–Cy5-labeled dsDNA and hybrid RNA/DNA duplexes as a function of duplex length. Due to the rigidly stacked dyes on the duplex ends and the double-helix structure of the duplexes, EFRET shows distance (1/rDA6) and orientation (changing κ2) dependence, resulting in a decreasing “bouncing ball” type curve, which is different for B-form dsDNA and A-form hybrid RNA/DNA. Reproduced with permission from ref. 155 Copyright 2008 by The National Academy of Sciences. (C) DNA with firmly incorporated fluorescent base analogues as D and A shows a similar “bouncing ball” type EFRET distance-orientation dependence due to the DNA double-helix structure. Reproduced with permission from ref. 156 Copyright 2017 American Chemical Society. (D) EFRET for Tb-Cy5.5-labeled dsDNA, and PL lifetimes of Tb (green open squares) and Cy5.5 (magenta dots) as a function of duplex length. Due to the long excited-state lifetime of Tb (∼2.7 ms) and its isotropic emission, FRET in the DNA double helix is only distance dependent and dynamic averaging with a constant orientation factor (κ2 = 2/3) becomes applicable. Significant uncertainties become only visible for very long D–A distances beyond 9 nm. Reproduced with permission from ref. 157 Copyright, 2017, John Wiley & Sons.
