Analyses of RNA–Ligand Interactions by Fluorescence Anisotropy - Biophysical Approaches to Translational Control of Gene Expression - page 177

Many of these screens are based on competition assays (Owicki 2000 ) . By this method,
a small fluorescent ligand associates with a large macromolecule resulting in a high
anisotropy value owing to the large molecular volume of the complex containing the
fluorophore. When added to the complex, a chemical inhibitor of this interaction
would be expected to induce release of the fluorescent substrate, which is detected by
a significant decrease in the measured anisotropy of the binding reaction.
For a screen that may encompass thousands or even hundreds of thousands of
binding reactions, determining the effectiveness of the assay design is critical if
meaningful data are to be obtained. The Z -factor was introduced to address this need
(Zhang et al. 1999 ) .

3 ss + 3

S C

Z =− 1 (9.10)

| mm − |

S C

Here, s is the standard deviation of the samples and m is the mean. The subscripts
S and C designate sample and control, respectively. When values of Z are close to 1,
the means of the control and sample are well-separated with little variation between
data points, signifying that the assay is designed appropriately to identify individual
compounds (hits) that significantly perturb reaction binding equilibrium. Conversely,
if the Z -factor is close to 0, essentially no difference can be identified between
samples and negative controls.
In addition to high-throughput screening, using plate readers to measure
fluorescence anisotropy is appealing because they can accommodate very small
sample volumes (often down to 100 mL in 96-well mode, and even smaller in 384-
well mode) (Mao et al. 2006 ). This feature can be particularly valuable when the
components of the model system are expensive or difficult to generate. Furthermore,
current generations of fluorescence anisotropy-capable plate readers are generally
user friendly, require limited optimization, and often include many other detection
modes. However, plate readers also suffer from some limitations with respect to
measuring anisotropy. They are generally not as sensitive as cuvette-based
spectrofluorometers and offer less flexibility in probe selection since excitation and
emission wavelengths and bandpass limits are normally set using optical filters
rather than monochromators. In addition, plate readers are not as useful for evalua-
tion of pre-steady-state kinetics, because they are not compatible with the stopped-
flow mixers necessary to initiate very rapid biochemical reactions.

9.3 Data Analysis

9.3.1 Additivity of Anisotropy

The most common applications of fluorescence anisotropy to the study of RNA-
protein binding events are to quantitatively measure the affinity of these interactions.