Analyses of RNA–Ligand Interactions by Fluorescence Anisotropy - Biophysical Approaches to Translational Control of Gene Expression - page 169

Chapter 9
Analyses of RNA-Ligand Interactions
by Fluorescence Anisotropy

Aparna Kishor , Gary Brewer , and Gerald M. Wilson

9.1 Introduction

The use of fluorescence anisotropy-based techniques for the accurate, quantitative
determination of biomolecular binding parameters has become increasingly common.
Applications span a wide range of biological disciplines, from the fields of medical
diagnostics and drug discovery to basic research. Some advantages of this tech-
nique, among others that will be discussed in Sect. 9.4 , are that it does not require
radioactive probes and that it is a homogenous-phase assay. Parameters including
equilibrium binding constants, binding site size, and Gibb's free energy can be cal-
culated from binding isotherms generated under various temperature and probe con-
ditions. Additionally, monitoring fluorescence anisotropy under pre-steady-state
conditions can resolve on- or off-rates of binding. For equilibrium binding experi-
ments, data collection can often be streamlined by using plate readers configured to
measure fluorescence anisotropy, several of which are commercially available.
Traditional format fluorescence spectrophotometers equipped with polarizers,
however, are still common; although these typically can only read one sample at a
time, their sensitivity is generally superior to plate readers. Furthermore, single-cell
spectrofluorometers are normally more versatile, permitting, for example, measure-
ments under pre-steady-state conditions or across gradients of temperature.
The basic principle behind the use of anisotropy to measure macromolecular
binding parameters is that the mobility of a molecule in solution will change when it