Background

Fluorescence polarization anisotropy has become one of the most popular methods to measure the interaction of proteins with a large variety of ligands including small molecules, nucleic acids, peptides and other proteins. The method is quick, inexpensive and can be modified to be used in plate readers equipped with fluorescence detectors. The technique is based on the principle that when a fluorescent molecule is excited with plane polarized light, the emitted light remains polarized in the same plane if the molecule is stationary or if it rotates slowly. In contrast, if the molecule rotates rapidly (due to small size), the light is emitted in a different plane. These changes can be quantified by the normalized differences in parallel and perpendicular intensities. Polarization is defined as P = (I₌ - I_⊥)/(I₌ + I_⊥), where I₌ is the parallel intensity and I_⊥ is the perpendicular intensity. An alternative way is to define the anisotropy, A = (I₌ - I_⊥)/(I₌ + 2I_⊥). Both parameters can be used interchangeably to describe the changes in polarization. Thus, when a small fluorescent DNA molecule binds a protein, the larger complex will rotate more slowly than the DNA molecule, changing the plane of the polarized light and increasing the anisotropy value. We have used this technique to measure the binding affinity of AAV Rep68 for different DNA substrates (Yoon-Robarts et al., 2004; Musayev et al., 2015; Bardelli et al., 2016). The non-structural AAV Rep proteins carry out most of the DNA transactions that are required to complete the virus life cycle. These include DNA replication, transcriptional regulation, site-specific integration and packaging of DNA into preformed capsids (Im and Muzyczka, 1990; Weitzman et al., 1994; Wonderling et al., 1995). The large AAV Rep proteins (Rep78/Rep68) contain an N-terminal origin binding domain (OBD) that specifically binds the Rep binding sites (RBS) and displays nuclease activity (Hickman et al., 2004; Musayev et al., 2015). The RBS sites consist of two or more 5’-GCTC-3’ repeats and are found at the viral origin of replication, in several promoters and at the AAVS1 integration site (Weitzman et al., 1994; McCarty et al., 1994). In addition, a C-terminus SF3 helicase domain is required for high affinity binding and DNA unwinding (James et al., 2003; Mansilla-Soto et al., 2009).The protocol described here can be modified to fit any protein-DNA system or any other instrument such as plate readers.