I am trying to design a DNA probe containing both a fluorophore and a quencher. I need it far enough (30 to 45 bases apart; 10-15 nm), but i still need the fluorescence to be quenched.
I know it greatly depends on the pair of fluorophore and quencher used. But it's quite expensive to buy probes with both of them modified, perhaps some can share their experience of trying this out? Thank you very much..
Would greatly appreciate these info:
-what fluorophore-quencher pair did you use?
-how many bases apart did you try?
-how efficient is the quenching? (can be qualitative too, in case you didnt quantify)
(My personal longest is ATTO647 - Iowa Black Quencher; 20 nt apart.. qualitatively, still very good quenching observed. Anyone tried longer?)
Thanks!
The highest value of Ro that I have seen is about 70 Angstrom (7 nm), which means that if the distance between the donor and acceptor were 7 nm, the energy transfer efficiency would be 50% between one donor and one acceptor. Since FRET efficiency falls off very strongly with distance, the efficiency would be much lower at 10-15 nm. You could improve on that, in principle, by having more than one acceptor per donor, if you could work out the synthesis.
Here are a few tables of Ro values.
https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/tables/r0-values-for-some-alexa-fluor-dyes.html
https://www.atto-tec.com/fileadmin/user_upload/Katalog_Flyer_Support/R_0_-Tabelle_2018_web.pdf
You could try
Article Lanthanide Complexes and Quantum Dots: A Bright Wedding for ...
FRET transfer up to 10 nm are reported. But QD are bulky chromophores that can strongly portable the conformation of your DNA.
In case of QD, it will be SET not FRET, I feel. In that case distance will be more, nearly 300 A.
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