Biology Reference
In-Depth Information
short lifetime. More recently, the introduction of a mutated version of the Rluc
enzyme, the Rluc8 variant (Rluc mutant containing eight amino acid substitutions),
leads to an
5-30-fold increase in the original BRET 2 signal, which results in a
new BRET form, the so-called enhanced BRET 2 (eBRET 2 )( Kocan et al., 2008 ). In
the eBRET 2 , we can combine the advantage of greater spectral resolution of the orig-
inal BRET 2 with a higher quantum yield when using Rluc8. The introduction of a third
Rluc substrate, called EnduRen, the protected form of the coelenterazine, resulted in
aformofBRET 1 , known as extended BRET (eBRET) ( Pfleger et al., 2006 ). This
enables real-time monitoring of receptor-receptor interactions for extended periods
of time and provides stability over time that is logistically advantageous for high
throughput screening applications. The eBRET name can result in a nomenclature
confusion with the previous name eBRET 2 .
A similar confusion arises with the introduction of the BRET 3 . For some authors,
BRET 3 refers to a BRET form that results from a combination of a red-shifted fluor-
ophore (e.g., mOrange) with Rluc8, using as substrate EnduRen, although the system
was validated with coelenterazine h ( De, Ray, Loening, & Gambhir, 2009 ). How-
ever, for other authors ( Bacart et al., 2008 ), BRET 3 refers to a donor/acceptor pair
formed by a firefly luciferase (from Photinus pyralis) and acceptors whose excitation
peaks overlap with the emitted light at 565 nm (for instance, the 24-kDa DsRed fluo-
rescent protein; peptides labeled with Cy3 or Cy3.5) using as a substrate the D -lucif-
erin developed by Gammon, Villalobos, Roshal, Samrakandi, and Piwnica-Worms
(2009) . The firefly luciferase in BRET 3 shows lower cellular autofluorescence at
the emission wavelength (565 nm) and a more sustained light emission by firefly lu-
ciferase compared to Rluc. However, disadvantages are weak signals and overlap
between donor and acceptor emission peaks. In addition, the tendency of DsRed
to oligomerize has to be considered in these BRET experiments for proper data anal-
ysis. It is therefore highly recommended to use instead a DsRed-monomeric variant
(Clontech, USA).
Finally, a new BRET version has been introduced: the quantum dot-BRET (QD-
BRET) ( Bacart et al., 2008; Xing et al., 2008 ). QDs are semiconductor nanocrystals
excited at any wavelength ranging from UV to 530 nm, and their light emission
wavelength, which depends on their diameter, can cover the spectrum from blue
to near infrared. They are then suitable energy acceptors for BRET 1 - and BRET 2 -
based assays as their broad interval of excitation wavelengths overlaps the currently
used luciferase-emitted light. It should be mentioned that based on the work of
Medintz and Mattoussi, it seems that QDs are not so good as energy acceptor but
rather used so far as energy donor ( Medintz & Mattoussi, 2009 ). So far, QDs have
only been used in BRET 1 -based assays, where QDs, directly linked to Rluc, were
injected into mice and energy transfer monitored in the presence of coelenterazine
h( Xing et al., 2008 ). Even though not really useable at this time, the self-illuminating
feature of QD-BRET makes imaging technically possible and could be optimized in
the future to work in conditions where the generation of photon is limited such as in
tissues. The emission peaks are clearly separated, which makes QD-BRET ideal for
screening applications. But
its major disadvantages are the large size of QD
Search WWH ::




Custom Search