Dual FRET molecular beacons for mRNA detection in living cells.
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.
Santangelo
Philip J
PJ
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Nix
Brent
B
Tsourkas
Andrew
A
Bao
Gang
G
D001665:Binding Sites; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation* / Q000379:methods*; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D051034:Inhibitor of Apoptosis Proteins; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000235:genetics; D015335:Molecular Probes / Q000032:analysis* / Q000235:genetics; D009363:Neoplasm Proteins; D034443:RNA Transport; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012996:Solutions; D000077022:Survivin
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D015335:Molecular Probes; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012996:Solutions; D000077022:Survivin