The fluorophores CY3 and CY5 are often used as Acceptor-Donor pairs in FRET expe
ID: 203145 • Letter: T
Question
The fluorophores CY3 and CY5 are often used as Acceptor-Donor pairs in FRET experiments. Find on the Web who is the donor and who is the acceptor, what is the donor fluorescence lifetime and the Förster radius of the pair. In a DNA beacon these fluorophores are labeling the extremities of a 25 bps SsDNA molecule that can self hybridize to form a hairpin. Find the change in FRET efficiency between the open and closed conformation of the beacon (assume that in the open conformation the distance between the molecule's ends is that of a 25bps dsDNA).Explanation / Answer
Answer
The 516 nm excitation of the Cy3 component and Cy5 fluorescence emission at 663 nm.
So Donor is - Cy3
The fluorescence lifetime for the Cy3 donor is 0.15 ± 0.03 ns. The distance 4nm.
In the open conformation Cy3 present in one end say 5' of ssDNA and the Cy5 present in other end 3' of ssDNA. This is separated by long distance. so FRET not possible.
In the closed conformation dur to hair pin loop the 5' end and 3' end come closer together. The Cy3 and Cy5 are in foster radius so if excitation at 516 nm excite the Cy3 compound and it further excite the CY5 and it release the emission wavelength at 663nm in red color. The FRET efficiency is increased in closed conformation.