Analyses of RNA–Ligand Interactions by Fluorescence Anisotropy - Biophysical Approaches to Translational Control of Gene Expression - page 192

(Mao et al. 2006 ). First, binding of long fluorescent RNA substrates (95 nt or more,
MW > 30 kDa) can often be well resolved for even relatively small proteins (70 kDa),
although larger differences between protein and ligand size are still preferred, since
the local segmental motions within a long RNA strand will be better restricted by a
more massive binding partner. Second, RNA-protein binding reactions can often be
designed such that they are minimally impacted by modest concentrations of sol-
vents commonly used for drug libraries (e.g., DMSO, methanol). Finally, improve-
ments in the sensitivity and resolution of microplate readers now permit reproducible
fluorescence anisotropy readings from samples as small as 5 mL containing as little
as 1 nM of a fluorescent ligand. In fact, smaller sample volumes can improve the
precision of anisotropy measurements because they minimize the optical path
through the sample, which reduces the contributions of library compounds to opti-
cal density (Owicki 2000 ). The constantly evolving nature of microplate reader
technology, including the development of more sophisticated optics (Owicki 2000 ) ,
is continually improving their utility.
Often, when screening a large number of compounds for inhibition of protein-
RNA interactions, the mechanism by which the compound disrupts the complex is
initially less important to the investigator than the knowledge that it has an effect at
all. However, downstream validation and drug characterization studies can reveal
novel and unexpected mechanisms for inhibiting RNA-protein association. For
example, a fluorescence anisotropy-based HTS survey for drugs that could inhibit
ARE binding by the mRNA-stabilizing factor HuR identified several potent lead
compounds. Subsequent mechanistic studies applying a wide range of potential
binding models revealed that some compounds blocked HuR binding by preventing
protein dimerization that may be required for RNA binding, while another bound
HuR directly (Meisner et al. 2007 ). It remains to be seen whether these compounds
or their derivatives will ultimately be useful for suppressing expression of ARE-
containing mRNAs in vivo or have therapeutic utility in patients.

9.6 Conclusions

The quantitative characterization of protein-RNA interactions in vitro can provide
critical validation for the functional significance of putative RNA-binding trans -
regulatory proteins identified using cellular or genetic assays. Reciprocally, resolv-
ing binding mechanisms and RNA substrate requirements using in vitro approaches
can help direct downstream cellular studies, including screens for novel RNA tar-
gets, drug design, and the interface between RNA recognition mechanisms and cel-
lular signaling pathways. Fluorescence anisotropy-based binding assays have proven
to be a sensitive and flexible platform for rigorous quantitative biochemical analyses
of ribonucleoprotein complex formation and are revealing features of RNA-protein
interactions that are not resolvable by conventional biochemical approaches.