FRET sensors

Fluorescence or Forster resonance energy transfer (FRET) is widely accepted as being one of the most useful methods to observe biochemical events in vitro and in living cells. Generally, there are two forms of FRET sensors those based on a pair of genetically encoded fluorophores, usually employing fluorescent proteins from jellyfish or corals, or those based on small molecules that make use of small organic fluorophores. 

As we have seen above, FRET is a technique that provides precise information about distances between 10 and 100 A which is in the range of the size of biological molecules and proteins. Researchers have taken advantage of this feature and developed different strategies to synthesize FRET sensors that are able to follow in real time and with high sensitivity very diverse processes such as enzymatic activity, conformational change, or molecule-molecule interaction. The design of these FRET sensors is described below. 

Fig. 6.13. Different designs of FRET sensors. (A) Substrates for hydrolytic enzymes. (B) Sensors for bond formation. (C) Sensors based on conformational or structural change. (D) Environmentally sensitive probes. (E) Quenched activity-based probe to monitor small molecule-enzyme interaction. (F) Small molecule-enzyme interaction using a labeled protein.

Small molecule-based FRET sensors and their applications... 

Although there is now a limited set of FRET sensors available, the gap between the demand in biology and the development of new reporters is not closing. Especially small molecule-based sensors are... 

Fig. 6.22. Folate-FRET sensor structure and its application to measure disulfide bond reduction in endosomes. The molecule contains the folate moiety which is recognized by the folate receptor situated at the plasma membrane. This recognition leads to endocytosis and after some time to cleavage of the probe. 

Kikuchi, K. (2004). Recent advances in the design of small molecule-based FRET sensors for cell biology. Trends Anal. Chem. 23, 407-415. 

Ratio imaging is particularly suited for single-polypeptide FRET sensors. In these constructs FRET changes are due to altered distance and/or orientation of the donor and acceptor, and since the fluorophores are tethered their stoichiometry is always fixed. Thus, the filterFRET problems are easier to address and, assuming full maturation of both FPs [4], it can in fact be shown that under these circumstances two images suffice to calculate FRET quantitatively (see Textbox 1 and Appendix 7.A.6). 

In general, ratio imaging is not quantitative nor is it, strictly spoken, normalized because the acquired data do not permit Problems 1-4 (see Sect. 7.1.1) to be properly addressed. One important exception is the case where donors and acceptors are present at a fixed stoichiometry. Examples of that are the popular single-polypeptide FRET sensors. In this case, the normalization problem (2) is inherently solved and the overlap- and reference-image problems (1 and 3) simplify considerably. It can be shown [1 and Appendix 7.A.6] that in that case FRET efficiency ( ) can be calculated from D and S images. 

Looger, L. L., Lalonde, S. and Frommer, W. B. (2005). Genetically encoded FRET sensors for visualizing metabolites with subcellular resolution in living cells. Plant Physiol. 138, 555-7. 

Bogner, M. and Ludewig, U. (2007). Visualization of arginine influx into plant cells using a specific FRET-sensor. J. Fluoresc. 17, 350-60. 

Niino Y, Hotta K, OkA K (2009) Simultaneous live cell imaging using dual FRET sensors with a single excitation light. PLoS One 4 e6036... 

Amplification of C emission upon excitation of CPE, relative to that upon direct excitation of C is an important advantage of CPE-based FRET sensors, which benefits from the rapid intrachain and interchain energy migration from CPE to C via FRET. The detection sensitivity of CCP-based DNA sensor thus is enhanced to an extent dependent on the signal amplification of C emission. Amplification factor is defined as the intensity ratio of the saturated CCP-sensitized C emission to the intrinsic C emission in the absence of CCP. To acquire large signal amplification, it is necessary to review the factors affecting the FRET process from CCP to C. Equation (1) describes the calculation of FRET rate (KVRi T) [67] ... 

Replacing CFP with FlAsH in a FRET Sensor of G-protein Coupled Receptor (GPCR) Activation... 

One of the most successful and novel applications of GFP has been in the design of FRET sensors of biochemical pathways [1], Two-color mutants of... 

Fig. 22 Schematic illustration of nonexclusive QD/GNR based FRET sensors for chiral assays in two individual systems. Reprinted with permission from Xia et Copyright 2012 American Chemical Society.

A lot of work has been done on FRET sensors for DNA detection based on electrostatic and hydrophobic interactions between DNA and CPs [171-179]. FRET... 

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