Analyses of RNA–Ligand Interactions by Fluorescence Anisotropy - Biophysical Approaches to Translational Control of Gene Expression - page 173

Thus, temperature (in Kelvin) is inversely proportional to r . The rotational correlation
time ( q ) is a related value which is also commonly found in the literature. The two
values convey the same information as r = 3 q .
The Perrin equation relates observed anisotropy/polarization to the excited state
lifetime (the time between excitation and emission) and the rotational diffusion of a
fl uorophore.

A

0
3

A = (9.5)

⎛⎞
+ ⎜ ⎝⎠

t
r

1

where A is observed anisotropy, A 0 is intrinsic anisotropy, and t is the excited state
lifetime. Substituting r into the Perrin equation, we get

1 1 RT
AA AV

t

(9.6)

=+
0

h

0

The intrinsic anisotropy of a system does not normally vary unless the excitation
energy is transferred rather than emitted. This is commonly observed with Förster
Resonance Energy Transfer (FRET), where large angles between the absorption and
emission dipoles (on the FRET donor and acceptor fluorophores, respectively) can
significantly decrease measured anisotropy (Jameson and Ross 2010 ) . In addition,
FRET decreases the excited state lifetime of a fluorophore, but the lifetime can also
be impacted by the probe microenvironment (pH, hydrophobicity, etc.). However, if
the experimental design allows T , h , t , and A 0 to be held constant, then A will be
determined solely by changes in the effective molar volume ( V ). The size of the rotat-
ing molecule may be altered by aggregation, degradation, or, in the cases discussed
below, association/dissociation with a specific binding partner (Jameson and Ross
2010 ). However, there are also a few possible sources of error for A related to sample
composition. For example, scattered excitation light (caused by particulate matter or
large aggregates in a sample) is vertically polarized and will increase the apparent
polarization if not corrected (Owicki 2000 ; Lakowicz 1999 ) . Additionally, scatter
from emission is possible in samples with high optical densities, which will lower
observed polarization/anisotropy values (Owicki 2000 ; Jameson and Ross 2010 ) .

9.2.2 Considerations Relating to the Fluorophore

Spectral properties of a fluorophore important for fluorescence applications are
intrinsic polarization, lifetime, emission wavelength, and quantum yield (Yan and
Marriott 2003 ). There are a wide variety of fluorophores that may be conjugated to
RNA substrates and are compatible with anisotropy assays. However, selection of a
fluorophore with a lifetime appropriate for monitoring substrate motion in the given
assay conditions is essential (Jameson and Ross 2010 ) . Many commonly used
fluorophores like fluoresceins and rhodamines have fluorescence lifetimes in the low