Experimental and simulated fluorescence depolarization due to energy transfer as tools to study DNA-dye interactions

Abstract

A method to study DNA‐dye complexes by the combination of steady state fluorescence anisotropy measurements and computer simulations of the fluorescence depolarization due to resonance energy transfer is presented. The simulations are based on a Markov chain analysis, assuming random distribution of the dyes along the DNA chain and energy transfer that obeys Förster kinetics. Since the investigated intercalators (ethidium bromide, YO, PO) and groove binders [4′6‐diamidino‐2‐phenylindole (DAPI)] were found to show different depolarization dependence on binding density, the method can be used to quite sensitively characterize the binding mode. Excellent agreement between the measured and simulated anisotropy is found for all investigated intercalators. The proposed method gives an estimation of the unwinding angle for intercalators and provides information about the binding site size, and the presence or absence of sequence specificity. For the groove binder DAPI interacting with mixed sequenced DNA, the measured and computed depolarization do not agree, and this can be rationalized in terms of the high sequence specificity of this dye. However, for DAPI bound to [poly(dA‐dT)]2 the measured data agree well with computed data for a groove binder that is displaced a distance 7 Å from the helix axis and has a binding site size of three bases. © 1997 John Wiley & Sons, Inc. Biopoly 41: 481–494, 1997

A polarized view on DNA under tension

“…15 This angle was determined to be 51 • , 15 based on one bis-intercalating dye dimer sandwiched two base pairs and every chromophore unwinding the DNA helix by ∼20 • . 8,11,15,43 In the case of energy transfer between the chromophores, the emitted photon is equally likely emitted by either of the two. 15 Therefore, such energy transfer can be emulated in the simulations by setting β = 51 • /2 = 25 • as the effective azimuthal angle between the absorbing and emitting dipoles.…”

“…8,11,15,43 In the case of energy transfer between the chromophores, the emitted photon is equally likely emitted by either of the two. 15 Therefore, such energy transfer can be emulated in the simulations by setting β = 51 • /2 = 25 • as the effective azimuthal angle between the absorbing and emitting dipoles. In practice, the two chromophores of a YOYO dimer are indeed close enough such as to allow efficient transfer.…”

“…Polarization anisotropy has been used to study reorientations of DNA conformations both in steady state 15,16 and in a time-resolved manner, 17 for quantitation of rotational diffusion. Linear-dichroism (LD) measurements, on the other hand, probe the anisotropic absorption of linearly polarized light.…”

Electrophoresis with Polarized Fluorescence Detection. Application to Capillary Fluorescence Rejection

“…The simulation of Carlsson and co-workers show that DNA intercalated at 0.2 dye/bp caused depolarization 0.4. 11 Since the anisotropy data of the 1 kpb ladder samples at zero intercalation are unknown, the anisotropy calculations from Carlsson's model cannot be accomplished. Comparison with the data in Fig.…”

Two-photon fluorescence: Resonance energy transfer

“…Other multiphoton fluorescence studies that have been reported relate directly to biological and other systems featuring both single center chromophores and energy trapping sites. [10][11][12][13][14][15][16][17][18][19][20] These, together with the well-known use of FRET as a spectroscopic ruler, [21][22][23][24][25] reflect the practical utility of the theory to be developed.…”