Resonance Energy Transfer (RET)

 

Resonance energy transfer (RET) is a process where energy from a luminescent molecule (the donor) that is in its electronically excited state is transferred  to an acceptor molecule by dipole-dipole interaction. RET occurs when donor and acceptor molecules are in close proximity (usually within 10 to 80 angstroms). The acceptor molecule can either be another fluorescent dye or a nonfluorescent molecule. There are several parameters that play an important role for RET to occur: 

 

1)The absorption spectrum of the acceptor molecule must overlap the emission spectrum of the donor characterized by the Overlap Integral (J).

2) The Orientation between the donor and acceptor molecule plays only a role for fixed donor and acceptor pairs, for free rotating donor acceptor pairs it can be considered a constant.

3) The Quantum Yield of the Donor and the Extinction Coefficient of the Acceptor.

 

 

 

Figure 1. Plot showing the Energy Transfer between a Square-635 (donor)-labeled antibody and a Square-670 (acceptor)-labeled antigen. With increasing amounts of the antigen the donor fluorescence decreases and at the same time the acceptor fluorescence increases

Another characteristic parameter for a given energy transfer pair is the so called Förster Radius R0. It is defined as the distance at which energy transfer is 50% efficient. RET efficiency drops dramatically as the distance between donor and acceptor exceeds the Förster radius, decreasing by the inverse sixth power of the distance between the donor and acceptor. Examples of  values for the Förster radius of commonly used RET donor-acceptor pairs including the Square-635/Square-670 energy transfer pair are listed in Table 1.

 

 

Table 1. Dyes for Energy Transfer Pairs

 

Donor

Acceptor

 Förster Radius (Å)

Fluorescein

Tetramethylrhodamine

55

EDANS

Fluorescein

43

Cy3

Cy5

55

Square-635

Square-670

70 

 

 

RET is highly efficient if the donor and acceptor are positioned within the Förster radius, which is typically 30-60 angstroms. RET-based assays are commonly used in automated real-time assays of hybridization and PCR monitoring. RET-based probes can also be used to monitor DNA interactions in living cells. RET can be utilized in any application where the proximity of two conjugatable molecules is of interest. These include:

 

 

 
 

References

  
 

Oswald B., Lehmann F., Simon L., Terpetschnig E., Wolfbeis O.S., Anal. Biochem., 280, 272–277 (2000). 

  
 

Oswald B., Patsenker L., Duschl J., Szmacinski H., Wolfbeis O.S., Terpetschnig E. Bioconjugate Chem., 10, 925–931 (1999).