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Dual FRET molecular beacons for mRNA detection in living cells
Open accessAbstract
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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Cites
Visualization of Single RNA Transcripts in Situ
Femino, A. M., Fay, F. S., Fogarty, K., & Singer, R. H. (1998) · Science · doi:10.1126/science.280.5363.585 · Cited by 1,504
Localization and translation of mRNA in dentrites and axons
Job, C., & Eberwine, J. (2001) · Nature Reviews Neuroscience · doi:10.1038/35104069 · Cited by 202
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Intracellular Delivery by Membrane Disruption: Mechanisms, Strategies, and Concepts
Stewart, M. P., Langer, R., & Jensen, K. F. (2018) · Chemical Reviews · doi:10.1021/acs.chemrev.7b00678 · Cited by 747
Nano-Flares: Probes for Transfection and mRNA Detection in Living Cells
Seferos, D. S., Giljohann, D. A., Hill, H. D., Prigodich, A. E., & Mirkin, C. A. (2007) · Journal of the American Chemical Society · doi:10.1021/ja0776529 · Cited by 710
Ke, G., Wang, C., Ge, Y., Zheng, N., Zhu, Z., & Yang, C. J. (2012) · Journal of the American Chemical Society · doi:10.1021/ja3082439 · Cited by 196
Metadata
- Authors
- Santangelo, P. J.
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2004-03-26
- Volume
- 32
- Issue
- 6
- Pages
- e57-e57
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- OpenAlex
- W2147178517
- Cited by
- 373
- References
- 2
- FWCI
- 6.23
- Type
- journal article
- Language
- en
- Open access
- bronze
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