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Published online 14 April 2004

Nucleic Acids Research, 2004, Vol. 32, No. 6 e57
© 2004 Oxford University Press

Dual FRET molecular beacons for mRNA detection in living cells

Philip J. Santangelo, Brent Nix, Andrew Tsourkas and Gang Bao*

Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA

*To whom correspondence should be addressed. Tel: +1 404 385 0373; Fax: +1 404 894 4243; Email: gang.bao{at}bme.gatech.edu

Received December 24, 2003; Revised and Accepted March 24, 2004

The ability to visualize in real-time the expression level and localization of specific endogenous RNAs in living cells can offer tremendous opportunities for biological and disease studies. Here we demonstrate such a capability using a pair of molecular beacons, one with a donor and the other with an acceptor fluorophore that hybridize to adjacent regions on the same mRNA target, resulting in fluorescence resonance energy transfer (FRET). Detection of the FRET signal significantly reduced false positives, leading to sensitive imaging of K-ras and survivin mRNAs in live HDF and MIAPaCa-2 cells. FRET detection gave a ratio of 2.25 of K-ras mRNA expression in stimulated and unstimulated HDF, comparable to the ratio of 1.95 using RT–PCR, and in contrast to the single-beacon result of 1.2. We further revealed intriguing details of K-ras and survivin mRNA localization in living cells. The dual FRET molecular beacons approach provides a novel technique for sensitive RNA detection and quantification in living cells.


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