FRET-based biosensors
Förster resonant energy transfer (FRET) is a radiationless mechanism of energy transfer from the excited state of the luminescent donor molecule to the acceptor molecule. The latter can be either a luminophore or a nonemitting chromophore. For FRET to occur there must be four conditions fulfilled:
1) the distance between the donor and acceptor fluorophores must be smaller than 10 nm,
2) the donor emission spectrum and the acceptor absorption spectrum must overlap,
3) the donor and acceptor transition dipole orientations must be approximately parallel, and
4) the fluorescence lifetime of the donor molecule must be of sufficient duration.
FRET-based biosensors are designed so that the presence of the analyte alters one of the above-mentioned conditions, e.g., the interaction with the analyte increases the distance between the donor and the acceptor; hence, decreasing the efficiency of FRET. FRET-based sensors are widely applied for bioanalytical measurements in different fields of research, both for in vitro assays and for in vivo monitoring in cellular research. The library of fluorescent proteins offers a huge collection of donor-acceptor pairs suitable for FRET experiments in cellular research. However, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) is still the most widely used donor-acceptor pair.
An advantage of FRET-based measurements is that they are ratiometric: both the donor and acceptor emission or excitation intensities are determined. Ratiometric measurement regime reduces the disturbing environmental effects and variations in probe concentration and excitation efficiency, thereby increasing the reliability of a FRET-based assay for characterization of interaction between biomolecules.
We have mostly applied FRET-based biosensors for the detection of changes in the intracellular concentration of cAMP (Figure 1). These biosensors comprise a pair of fluorescent proteins that are joined by a cAMP-sensitive linker. The linker contains a cAMP-binding motif of the exchange factor directly activated by cAMP (Epac). These FRET-based biosensors that we apply for our studies have been developed and kindly provided by prof. Kees Jalink or or by prof. Martin Lohse . We apply the BacMam system for the expression of the biosensor in mammalian cells. The cAMP biosensor enables us to measure the biological response to the binding of ligands to different GPCRs that affect the cAMP signalling pathway, e.g., melanocortin 1 receptors (Mazina et al. 2012), dopamine 1 receptor (Mazina et al. 2015), luteinizing hormone receptor (Mazina et al. 2015). Furthermore, we have developed a BacMam system for the caspase biosensor Casper-GR for measuring the development of apoptosis in live cells over 24 hours.
1) the distance between the donor and acceptor fluorophores must be smaller than 10 nm,
2) the donor emission spectrum and the acceptor absorption spectrum must overlap,
3) the donor and acceptor transition dipole orientations must be approximately parallel, and
4) the fluorescence lifetime of the donor molecule must be of sufficient duration.
FRET-based biosensors are designed so that the presence of the analyte alters one of the above-mentioned conditions, e.g., the interaction with the analyte increases the distance between the donor and the acceptor; hence, decreasing the efficiency of FRET. FRET-based sensors are widely applied for bioanalytical measurements in different fields of research, both for in vitro assays and for in vivo monitoring in cellular research. The library of fluorescent proteins offers a huge collection of donor-acceptor pairs suitable for FRET experiments in cellular research. However, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) is still the most widely used donor-acceptor pair.
An advantage of FRET-based measurements is that they are ratiometric: both the donor and acceptor emission or excitation intensities are determined. Ratiometric measurement regime reduces the disturbing environmental effects and variations in probe concentration and excitation efficiency, thereby increasing the reliability of a FRET-based assay for characterization of interaction between biomolecules.
We have mostly applied FRET-based biosensors for the detection of changes in the intracellular concentration of cAMP (Figure 1). These biosensors comprise a pair of fluorescent proteins that are joined by a cAMP-sensitive linker. The linker contains a cAMP-binding motif of the exchange factor directly activated by cAMP (Epac). These FRET-based biosensors that we apply for our studies have been developed and kindly provided by prof. Kees Jalink or or by prof. Martin Lohse . We apply the BacMam system for the expression of the biosensor in mammalian cells. The cAMP biosensor enables us to measure the biological response to the binding of ligands to different GPCRs that affect the cAMP signalling pathway, e.g., melanocortin 1 receptors (Mazina et al. 2012), dopamine 1 receptor (Mazina et al. 2015), luteinizing hormone receptor (Mazina et al. 2015). Furthermore, we have developed a BacMam system for the caspase biosensor Casper-GR for measuring the development of apoptosis in live cells over 24 hours.
Figure 1. The principle of the cAMP biosensor. The biosensor comprises of an analogue of CFP (donor) and an analouge of YFP (acceptor) that are joined by a cAMP-binding linker. If the concentration of cAMP in the cell is low, then cAMP is not bound to the biosensor. The conformation of the nonbound state of the biosensor enables FRET between the two fluorophores. Therefore, exciting the acceptor with light (at 427 nm) results in the emission from the donor (measured at 530 nm), whereas the emission from the acceptor (measured at 480 nm) is low. Upon increase of the intracellular concentration of cAMP, the biosensor is able to bind to cAMP. This binding event leads to the conformational change that does not favor FRET. Hence, the emission of the acceptor (measured at 480 nm) increases and the emission of the donor (measured at 530 nm) decreases.