Fluorescence Anisotropy Polarization

Fluorescence polarization anisotropy of a ligand and its complexes with DNA or RNA can be given by the following expression ... 

The broad field of nucleic acid structure and dynamics has undergone remarkable development during the past decade. Especially in regard to dynamics, modem fluorescence methods have yielded some of the most important advances. This chapter concerns primarily the application of time-resolved fluorescence techniques to study the dynamics of nucleic acid/dye complexes, and the inferences regarding rotational mobilities, deformation potentials, and alternate structures of nucleic acids that follow from such experiments. Emphasis is mainly on the use of time-resolved fluorescence polarization anisotropy (FPA), although results obtained using other techniques are also noted. This chapter is devoted mainly to free DNAs and tRNAs, but DNAs in nucleosomes, chromatin, viruses, and sperm are also briefly discussed. 

Figure 4.1. Time scales for rotational motions of long DNAs that contribute to the relaxation of the optical anisotropy r(t). Experimental methods used to study these motions in different time ranges are also indicated along with the authors and dates of some early work in each case. FPA, Fluorescence polarization anisotropy (Refs. 15, 18-20, and 87) TPD, transient photodichroism (Refs. 28 and 62) TEB, transient electric birefringence (Refs. 26 and 27) DDLS, depolarized dynamic light scattering (Ref. 116) TED, transient electric dichroism (Refs. 25, 115, and 130) Microscopy, time-resolved fluorescent microscopy (Ref. 176).

Thomas, J. C., Allison, S. A., Appellof, C. J., and Schurr, J. M. (1980). Torsion dynamics and depolarization of fluorescence of linear macromolecules. II. Fluorescence polarization anisotropy measurements of a clean viral phi 29 DNA. Biophys. Chem. 12, 177-188. 

Fluorescence polarization (anisotropy) version 2 (IMAP) Fluorophore labeled peptides bind to special detection beads coated with trivalent metal binding results in change in Brownian motion measured as with FP1 above Versatile without need for antibody Susceptible to compound interference peptide must be relatively small precludes use of protein substrates Turek-Etienne (2003a)... 

Figure 48. Comparison of experimental and simulated fluorescence polarization anisotropies for the S, + 789 cm 1 excitation of jet-cooled r-stilbene. The anisotropies include convolution effects associated with the finite excitation pulse width. The upper trace was calculated using the theoretical results of Ref. 49. The lower trace was obtained from experiment. The inset shows a simulated decay for this excitation band. 

Aptamers have a strong potential for multiplex sensing of proteins. Thus, a chip-based biosensor was developed for specific detection and quantification of cancer-associated proteins in complex biological mixtures using immobilized fluorescently labeled DNA and RNA aptamers. Fluorescence polarization anisotropy was used for solid- and solution-phase measurements of target protein binding [51]. 

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