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Finally, Komatsu et al. established highly sensitive FRET biosensor back-
bones. Their strategy completely abolished the dependence of the sensors on
the orientation of the two fluorophores. Indeed, prediction of the exact ori-
entation of the donor and the acceptor in an optimized sensor is rather dif-
ficult. 103,126 Instead, they optimized biosensor backbones relying entirely on
the distance between the fluorophores by modifying the linker between the
PAABD and the substrate. 127 Regular repetitions of the motif (SAGG) n
(where n is the number of repetitions (13-61)) and the 72 polyglycine
linker 128
were utilized and compared. 127
All generated backbones
highlighted that
long and flexible linkers
reduce the proportion of
biosensors folding in the basal state,
thereby improving the dynamic
range of biosensors.
6.4. Choosing a FRET pair
Historically, the first FRET measurements were performed with the BFP
and the enhanced green fluorescent protein (eGFP) as the FRET pair com-
bination. 1,129 Although these FPs meet the requirements for FRET
measurements, BFP has an unfortunate tendency to bleach much faster
than eGFP. The cyan fluorescent protein (CFP) and the yellow
fluorescent protein (YFP) FRET pair quickly replaced the BFP-eGFP
FRET pair to monitor Ca 2 þ variations in individual live cells. 130
The continuous improvement of fluorescent proteins, while beneficial
for biosensor optimization, could become overwhelming, as the possible
combinations seem endless. Nowadays, cloning procedures are much easier,
thus easing up the process. The choice of a FRET pair should be guided by
(1) up-to-date/optimized fluorophores, (2) which are red-shifted to mini-
mize phototoxicity, and (3) have monoexponential lifetimes when consid-
ering FRET-FLIM measurement methods. The idea here is to tap into the
distance ( R 0 ) and eventually the dipole-dipole orientation (k
2
) in order to
maximize FRET efficiency (see Section 2 )
6.4.1 Blue/yellow FRET pairs
This “original FRET pair” is still used in most biosensors today because of its
good spectral properties. Many variants derived from these FPs have rapidly
emerged and significantly increased FRET efficiency. The monomeric (m)
Cerulean 34 and the mTurquoise2 131 are the preferred variants of the CFP,
while monomeric Citrine and Venus 37 have proved to be the most popular
variants of the YFP. Cerulean has a better quantum yield, a higher extinction
coefficient, a fluorescence lifetime with a single-exponential decay, and an
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