Analyses of RNA–Ligand Interactions by Fluorescence Anisotropy - Biophysical Approaches to Translational Control of Gene Expression - page 185

binding constants from the ratio between binding on- and off-rates. For the induced-fit
binding mechanisms normally associated with biomolecular interactions, extraction
of multiple on- and off-rates (which include both intramolecular as well as intermo-
lecular events) from SPR data provides a significant challenge. By contrast, the
mathematical relationships relating fluorescence anisotropy to the fractional con-
centrations of fluorophore-tagged reactants and products are well established and
lend themselves to quantitative characterization using several different models of
complex formation (Wilson 2005 ) .
Several potential drawbacks to the application of anisotropy-based assays to
RNA-protein binding studies also exist. First, the RNA substrate must be fluorescent,
which requires covalent modification to incorporate the fluorescent dye. This intro-
duces the risk that the dye itself may contribute to or inhibit protein binding. This
possibility can be tested by comparing the K d value resolved using the fluorescent
RNA substrate with the K i value calculated from a competition experiment with the
unlabeled RNA ligand (Anderson et al. 2008 ). Second, for relatively weak binding
events (i.e., K d > 1 mM), generation of binding isotherms requires large amounts of
protein to approach saturation. Besides the logistical issues of producing and puri-
fying large quantities of some RNA-binding proteins, at high protein concentrations
sample viscosity is no longer constant and must be accounted for during data analy-
sis. One approach that can remedy this complication is to run a parallel binding
isotherm but using the dye alone as the substrate; provided the dye does not bind the
protein itself, protein-dependent changes in sample viscosity will be detected by
increases in probe anisotropy. Finally, synthetic RNA substrates containing
fluorescent labels remain fairly expensive (Mao et al. 2006 ). This cost can be com-
pounded in cases where several rounds of optimization are required, such as situa-
tions where several probes and/or linkage positions must be screened to circumvent
adverse interactions between dyes and binding proteins, or protein-dependent
changes in fluorescence quantum yield.

9.5 Applications of Fluorescence Anisotropy

In recent years, many groups have used fluorescence anisotropy to investigate vari-
ous biological pathways in which RNA and protein must interact. Below are a few
examples, along with some useful extensions of the technique.

9.5.1 Examples of RNA:Protein Complexes Analyzed

by Fluorescence Anisotropy

Targeted modification of an RNA substrate is a common strategy to identify
sequence and/or structural preferences of specific binding proteins and can also
provide insights into binding mechanisms. One study shows that Pumilio (Pum), an
RNA-binding protein that recognizes Nanos Response Elements (NREs) in the