From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 189

principle, comparative analysis of EKAR and Erkus revealed that the
dynamic response of EKAR was greatly improved; the dynamic range of
EKAR was higher than that of Erkus. 42
Details regarding FHA andWWdomains can be found in Refs. 120,121 ,
respectively.
6.3. Optimal linker combinations in FRET-based biosensors
Manipulating the linkers (i.e., swapping the linker from one sensor to another)
might prove to be an easier way and should be considered in the first instance.
Making highly sensitive FRET biosensors remains difficult and requires fine-
tuning. In spite of being time consuming, linker optimization is a crucial step
in biosensor design. In fact, FRET efficiency depends essentially on the distance
and the orientation of the two fluorophores (see Section 2 ), 122 which places
linkers at the heart of the dynamic range of biosensors. Linkers are mainly com-
posed of amino acids such as glycine, proline, and alanine, which gives them full
flexibility. The classical flexible linker consists of (GGSGGS) n which keeps
fluorophores at a “safe” distance fromone another, 123 and the rigid linker is com-
posed of (EAAAR) n where FPs are held in “fixed” distance and orientation.
In order to optimize and accelerate the development of FRET-based
biosensors, Ibraheem et al. 124 used a reliable high-throughput method by
undertaking the optimization of a methylation-sensitive H3K27 sensor—
H3K27-MetBio (trimethylation of lysine 27 of histone 3)—mainly based
on changes in the length of the linkers. They focused on the optimization
of the linker between the PAABD and the substrate. Screening of biosensor
variants was performed in colonies of Escherichia coli through the generation
of many hundreds of different linker combinations using several screening
libraries. The efficiency of H3K27-MetBio was improved, with a FRET
signal efficiency 2.3 times larger than the original sensor 124 .
In a recent study, Piljic et al. focused on the optimization of linkers
flanking the FPs. 125 They developed a reliable and rapid method to generate
multiple genetically encoded FRET sensor variants and tested them in re-
versely transfected mammalian cells. Long linkers improve the flexibility
of the sensor and favor orientation of the donor toward the acceptor fluo-
rophore, even though, sometimes, certain short and rigid linkers produce a
greater FRET signal. 125 Improvements of biosensors could also be achieved
by varying the linker composition in amino acids using combinatorial and
randomly generated linker libraries for detecting the most effective linker
composition.