Analyses of RNA–Ligand Interactions by Fluorescence Anisotropy - Biophysical Approaches to Translational Control of Gene Expression - page 174

nanosecond range, a scale similar to the tumbling motions of small macromolecules
in solution. Quantum yield is defined as the ratio between number of photons emit-
ted and number of photons absorbed. As such, assays using fluorescent dyes with
high quantum yields are more sensitive, permitting accurate quantitation at lower
probe concentrations. For monitoring tight binding equilibria where dissociation con-
stants ( K d ) are in the low nanomolar range or below, measurement of anisotropy at
sub-nanomolar substrate concentrations simplifies data analysis (described below)
and is thus best served by fluorescent tags of high quantum yield. Ideally, the quantum
yield of the selected fluorophore will not change as a result of interaction between
the RNA substrate and its cognate binding partners; management of this issue is
discussed further below.
Many proteins exhibit intrinsic fluorescence due to the presence of tryptophan,
tyrosine, or phenylalanine residues. Anisotropy based on measurements of intrinsic
protein fluorescence is possible and has been used in studies of protein-protein
interaction or time-resolved anisotropy (Beechem and Brand 1985 ), but is not com-
monly used when studying RNA-protein binding events. The popular fluorescent
proteins (GFP, etc.) are not generally useful for measurements of anisotropy in vitro
as the fluorescent components are largely immobilized within the framework of
these proteins and are thus relatively unresponsive to changes in molecular dynam-
ics (Yan and Marriott 2003 ). In quantitative analysis of RNA-protein interactions,
the RNA moiety is generally the smaller and more flexible binding partner. As a
result, in fluorescence anisotropy-based analyses of these binding events, the RNA
substrate is typically labeled with the fluorescent dye since this molecule normally
experiences the greater net restriction of conformational motion during formation of
the ribonucleoprotein complex. Most often, the fluorophore is located at one or the
other end of the oligonucleotide, but internal sites may also be used (Wilson 2005 ) .
Several commercial RNA synthesis services offer a variety of options for tagging
RNA oligonucleotides with these dyes. Fluorescein is popular for these applica-
tions, due to the fact that it is relatively inexpensive and that it can be readily conju-
gated. Challenges of using fluorescein are that it will suffer from photodegradation,
particularly in environments containing oxygen, and that its spectral properties are
pH-sensitive (Jameson and Ross 2010 ). The poor photostability of fluorescein is a
particular issue in kinetics experiments, where the anisotropy of a sample will be
read many times as a function of time. Other dyes that are commonly conjugated to
RNA substrates include 5¢-carboxytetramethylrhodamine (TAMRA) and the
AlexaFluor and cyanine families of dyes.

9.2.3 Considerations Relating to Instrumentation

9.2.3.1 Conventional Spectro fl uorometers

Fluorescence anisotropy is measured using a fluorescence spectrophotometer (also
called a spectrofluorometer) that is equipped with polarizers on both the excitation