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Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons
Published on: August 6, 2014
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Quantifying Gene Expression in Living Cells with Ratiometric Bimolecular Beacons
Yantao Yang1, Mingming Chen1,2,3, Christopher J Krueger1,4
1Department of Biomedical Engineering, College of Engineering, Peking University, No. 5 Yiheyuan Road, Haidian District, Beijing, 100871, China.
Methods in Molecular Biology (Clifton, N.J.)
|November 14, 2017
Summary
Molecular beacons (MBs) can enter cell nuclei, causing false signals. Ratiometric bimolecular beacons (RBMBs) were developed for improved cytoplasmic retention, enabling accurate single-molecule RNA quantification in live cells.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Molecular beacons (MBs) are oligonucleotide probes for studying RNA in live cells.
- MBs often localize to the nucleus, leading to false positive signals.
- Existing strategies to mitigate nuclear entry include backbone modifications or nuclear export facilitation.
Purpose of the Study:
- To develop a novel RNA imaging platform with enhanced cytoplasmic retention.
- To create ratiometric bimolecular beacons (RBMBs) by combining MBs and siRNA functionalities.
- To enable sensitive and accurate single-molecule quantification of mRNA transcripts.
Main Methods:
- Synthesis of ratiometric bimolecular beacons (RBMBs).
- Integration of siRNA-like functional elements for nuclear export.
- Application of RBMBs for live-cell imaging and RNA quantification.
Main Results:
- RBMBs demonstrated long-term cytoplasmic retention in living cells.
- RBMBs exhibited minimal false positive signals compared to traditional MBs.
- RBMBs accurately quantified engineered mRNA transcripts at the single-molecule level.
Conclusions:
- RBMBs represent a significant advancement for live-cell RNA imaging.
- The platform overcomes the nuclear sequestration issue of conventional MBs.
- RBMBs facilitate absolute quantification and tracking of single mRNA transcripts.
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