Fluorescence Technologies for Monitoring Interactions Between Biological Molecules In Vitro - Progress in Molecular Biology and Translational Science - page 117

1.3. Fluorescence for biological molecules
Over the last two decades, fluorescence spectroscopy technologies have been
extensively applied to understand specific molecular interactions in different
biological and biochemical pathways, including monitoring major cellular
events and enzyme mechanisms and sensing cellular response leading to aber-
rant behavior. Fluorescence approaches allow the characterization in a non-
invasive manner, at the steady- and pre-steady-state levels of protein/protein,
protein/nucleic acid, protein/substrate, andbiomembrane/biomolecule inter-
actions as well as enzymatic assays or competitive immunoassays, which con-
stitutes a major advantage over other methods used for this purpose. Although
fluorescence spectroscopy technologies provide valuable answers to numerous
questions concerning the identity, location, conformation, andenvironment of
any protein or nucleic acid, the sensitivity of the technologies also requires tak-
ing into account the limitations of the approach and keeping inmind that every
small change in a parameter may induce variations. 19 Thus each specific
approach has several advantages and limitations ( Table 4.1 ).

Table 4.1 Advantages and limitations of fluorescence techniques (adapted from Pope
et al.) 19
Technique Advantages Disadvantages and limitations

Fluorescence
intensity

- Simple - Little information for quality

control - Suitable for fluorigenic

assays - Sensitive to inner-filter and

autofluorescence interference - Readily miniaturized

Fluorescence
polarization/
anisotropy

- Simple and reasonably

predictive

- Local motion effects

-

Suitability limited by lifetime of
dye, ligand size, and molecular
weight change

- Insensitive to inner-filter

effects

- Ratiometric technique - Dynamic range limited

- Improved well-level

quality control

- Can suffer from autofluorescence

- Suitable for small ligands

( < 15 kDa)

Fluorescence
resonance
energy transfer
(FRET)

- Simple and reasonable

predictable

- Requires multiple lablels

-

Sensitive to inner-filter and
autofluorescence interference

- Suitable for short inter/

intramolecular distances
(

- Limited to short distances to

obtain high signal changes < 5 nm)

- Range of available

donors and acceptors

- Most dyes monitor only donor

quenching