BASICS OF FLUORESCENCE POLARIZATION

Fluorescence polarization (FP) is a commonly used tool for investigating molecular processes which involve interactions or fluidity. FP is a homogeneous technology and reactions are very rapid, taking seconds to minutes to reach equilibrium.  Because FP is a fundamental property of the molecule, the reagents are stable, and this results in a high level of reproducibility. FP has proven to be highly automatable, often performed with a single incubation and a single, premixed, tracer-receptor reagent. The fact that there are no washing steps increases the precision and speed over heterogeneous technologies and dramatically reduces waste.  FP is independent of fluorescence intensity, so this technology is unaffected by turbidity or the presence of dyes.  High-throughput screening is now accommodated with the advent of polarizing optics on microplate readers.

FP is a technique perfectly aligned with the study molecular interactions. It gives a direct, nearly instantaneous measure of a tracer`s bound/free ratio.  FP experiments are done in solution, allowing true equilibrium analysis down to the low picomolar range. FP measurements do not adulterate samples, so they can be treated and reanalyzed in order to ascertain the effect on binding by changes such as pH, temperature, and salt concentration.

FP measurements are based upon the assessment of the rotational motions of a molecule.  It is extremely efficient at measuring discreet differences in molecular mass which occur during large molecule/small molecule interactions, such as antibody/ligand or receptor/ligand binding.  In general, a dye is attached to a small molecule, or tracer.  It is then possible to measure its binding to a molecule of greater size, through its speed of rotation, in real-time or as an end-point assay.

Principle Behind Fluorescence Polarization: 

When a tracer molecule (fluorophore bound to some other molecule) is excited by polarized light, it will emit light in the same polarized plane, provided that the molecule remains stationary throughout the excited state. If the molecule rotates and tumbles out of this plane during the excited state, light is emitted in a different plane from the excitation light. If vertically polarized light is exciting the fluorophore, the intensity of the emitted light can be monitored in vertical and horizontal planes (degree of movement of emission intensity from vertical to horizontal plane is related to the mobility of the fluorescently labeled molecule). If a molecule is very large, little movement occurs during excitation and the emitted light remains highly polarized. If a molecule is small, rotation and tumbling is faster and the emitted light is depolarized relative to the excitation plane.  Large molecules tend to rotate slowly while small molecules rotate rapidly, as the rate of rotation is inversely proportional to a molecule's size.   Small molecules rotate quickly during the excited state, and upon emission, have low polarization values. Large molecules, caused by binding of a second molecule, rotate little during the excited state, and therefore have high polarization values.  This change in rotational speed is the essence of all FP assays.

Advantages of the Usage of Far Red Tracers:

FP is a robust technology free of many of the reaction condition restraints common to many HTS assay formats.  For example, turbidity, color quenching or the presence of colored dyes do not serve as obstacles to effective performance of FP assays. But with the sensitivity and simplicity of the FP assay format does come some forms of interference.

Compound Autofluorescence
Background fluorescence from drug library compounds can cause artifacts in the FP assay format.   The presence of background fluorescence can be addressed by pre-reading the fluorescence in a well before the addition of the fluorescent tracer. FP instruments are capable of then subtracting out background fluorescence before the calculation of the FP value. This background subtraction on individual wells is likely impossible during a primary screen, but can be performed on secondary screens of tagged compounds.
The vast majority of background fluorescence issues are encountered in the green fluorescence range of wavelengths (475-550 nm).  Most of these small molecules with intrinsic fluorescence will emit a depolarized signal (gives a low polarization value), thus it is likely that these compounds will give aberrant results (false positive or false negative results depending upon the assay format) in the FP assay format. Tracers in the far red wavelengths greatly decrease the likelihood of issues with background fluorescence interference.

In order for a molecule to absorb light in the far red wavelengths it must contain a high degree of adjacent double bonds and aromatic rings (conjugation).  The higher the level of conjugation, the higher the ability of a molecule to absorb light in the far red range (greater than 600 nm).  The high degree of conjugation necessary for “red” fluorescence is generally incompatible with most “druglike” properties, and therefore there are, in general, fewer “red” fluorescent compounds in most drug libraries.

Light Scatter
Like autofluorescence, scattered light can adversely affect FP assay condtions.  In general, light scatter arises from the presence of precipitant matter within the assay well.  This scattered light is generally highly polarized and this scattered light results in erroneously high polarization value. In many assay formats, this result would be read as a false positive “hit” in a compound library screen.  This “hit” would require additional test and resources to go back and determine that this positive reading was indeed an assay artifact.  Light scatter is more efficient in the green wavelength range and is less of an issue at higher wavelengths.

Summary
False positives and false negatives which occur during the process of compound library screening, increase the costs associated with “hit” determination.  The choice of tracers for FP in the far red wavelength range (>600 nm) has been shown to be a useful strategy to overcome interference due to autofluorescence or light scatter seen a lower wavelengths.  Published studies have shown an 84% reduction in fluorescent compounds when moving from wavelengths used for fluorescein-based tracers to wavelengths used by far red tracers. The cost savings expected in terms of time saved in follow-up screening of false positive or false negative results is substantial.

 

Helpful resources:

“Technical Resource Guide; Fluorescence Polarization – 4th Edition,” Invitrogen Corporation, 2006. 

Vedvik, KL et al: Overcoming Compound Interference in Fluorescence Polarization-Based Kinase Assays Using Far-Red Tracers. ASSAY and Drug Development Technologies 2(2):193-203; 2004.

Turconi S, Shea K, Ashman S, Fantom K, Earnshaw DL, Bingham RP, Haupts UM, Brown MJ, Pope AJ: Real experiences of uHTS: a prototypic 1536-well fluorescence anisotropy- based uHTS screen and application of well-level
quality control procedures. J Biomol Screen 2001;6: 275–290.

Checovich WJ, Bolger RE, Burke T: Fluorescence polarization—a new tool for cell and molecular biology. Nature 1995;375: 254–256.

Burke T, Bolger R, Parker G, Schall R: Kinase activity measurement using fluorescence polarization. Filed Oct. 28, 1997; Patent #: WO9818956A1: Published May 7, 1998.

Banks P, Gosselin M, Prystay L: Impact of a red-shifted dye label for high throughput fluorescence polarization assays of G protein-coupled receptors. J Biomol Screen 2000; 5:329–334.