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gives access to pairs with different intrinsic
R
0
while keeping the high signal-
to-noise ratio, thanks to the lanthanides donor. From a biological point of
view, it makes it possible to detect and followmolecular events or conforma-
tional changes occurring in cells at different amplitudes, with a better accuracy
as compared to classical FRET, in the range from 1 to 10 nm. A considerable
advantage used in the HTRF technology is the ratiometric approach where
the acceptor signal is divided by the donor signal to correct for compound in-
terference.
14
A final advantage is that lanthanide emission is unpolarized
because of its long excited-state lifetime. This phenomenon suppresses the
possibility that the acceptor and the lanthanide-cryptate dipole moments be-
come perpendicular to each other and thus prevents the eventuality of no
transfer of energy between them. This allows a more accurate interpretation
of the TR-FRET signals measured as compared to studies with classic FRET
signals, where this parameter is rarely taken into consideration.
2.4. TR-FRET in HTS campaigns
All these characteristics give TR-FRET strong advantages over other HTS
screening technologies and over other fluorescent techniques. First of all, its
combination of high sensitivity and robustness makes miniaturization pos-
sible, even up to a 1536-well plate format. It is a safe method as compared
to radioactive assays, and waste handling is not a major issue anymore. A re-
duced quantity of lanthanide-labeled entities is needed when compared to
classical fluorescent probes, thanks to the high signal-to-noise ratio, which
can yield important savings in an HTS campaign. And, nowadays, the va-
riety of assays and ligands available is continuously increasing, offering
TR-FRET solutions to assess almost all events occurring during activation
and signaling of a GPCR. A few drawbacks still remain with TR-FRET.
It is still less sensitive than radioactive assays, and it can be cumbersome
to develop TR-FRET-compatible fluorescent ligands that retain a good
affinity for the target. On the same aspect, the labeling of the target with
TR-FRET fluorophores is not always possible or easy, and in the case of
GPCRs, it is often necessary to modify the receptor, which could affect
its properties (this will be developed in part
4.1
and
4.2
). Finally, the
range of linearity for some quantitative assays is limited, and assays are often
more expensive than those based on genetically encoded fluorescent pro-
teins or classical fluorophores. However, when compared to the limitations
of other techniques, it is no surprise that TR-FRET assays are more and
more widely used.
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