You are designing a FRET experiment to measure the folding a

You are designing a FRET experiment to measure the folding and unfolding of a RNA molecule. You have labeled the two ends of the RNA molecule with Cy3 and Cy5 dye, respectively. The Forster distance is 5 nm for these two dyes. Assuming both fluorophores have the same brightness. You observed that the FRET ratio has jumped from 0.3 to 0.7 when you increases the temperature. What conclusion can you draw from this experiment? For the same experiment, you realized that you used an inappropriate dichroic filter to separate Cy3 and Cy5 fluorescence. 20% of Cy3 emission actually goes to the Cy5 detector. What is the new conclusion you have for your experiment?

Solution

Answer:

1) As the temperature increases, the energy on the RNA molecule increases. as a result folding of RNA occurs which increases the FRET ratio 0.3 to 0.7