Analyses of RNA–Ligand Interactions by Fluorescence Anisotropy - Biophysical Approaches to Translational Control of Gene Expression - page 170

Fig. 9.1 Conceptual schematic for the use of anisotropy to measure RNA binding. The green
sphere is the fluorescent tag. If the tagged molecule is single-stranded RNA, then both segmental
and global tumbling motions will contribute to fluorophore mobility in solution. Both of these
types of motion will be restricted upon binding to protein ( red ellipsoid)

binds another molecule (it should be noted that “anisotropy” as it appears throughout
this chapter is distinct from the physical chemistry term). In solutions of a given
viscosity, smaller molecules will tumble faster than larger ones (Fernandes 1998 ) .
The examples described in this chapter feature interactions between small RNA
substrates and the proteins that bind them. In this context where the RNA ligand is
highly mobile owing to its relatively small size and large potential for segmental
motion, its association with a protein or protein complex decreases its molecular
motion owing in part to the increased size of the ribonucleoprotein complex.
In order to measure the protein-dependent change in the mobility of an RNA
ligand, anisotropy-based assays typically use a fluorescently labeled RNA substrate
(Fig. 9.1 ). Upon exposure of this fluorophore to plane-polarized light, only molecules
that have their absorption transition dipole moment oriented appropriately will
become excited (Gradinaru et al. 2010 ). In the absence of molecular motion, all emis-
sion from these fluorophores would be observed in the same plane as excitation.
However, if the excited fluorophores are rapidly tumbling in solution during the
excited state lifetime, emitted light will be depolarized. By contrast, if mobility of the
excited fluorophores is limited (by association with a large macromolecular complex,
for example), emitted light will be more highly polarized, since fewer excited mole-
cules will tumble out of the plane of excitation before quantum emission (Fernandes
1998 ; Jameson and Ross 2010 ). The degree of emission depolarization is quantified
by parameters termed polarization or anisotropy , both of which are defined below.

9.2 Measuring Anisotropy

9.2.1 Theory

Passing an incident light beam through a polarizer limits transmission primarily to
light with an electric vector vibrating in a single plane. The direction of that vector