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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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Genetically Encoded Fluorescent RNA Sensor for Ratiometric Imaging of MicroRNA in Living Tumor Cells
Zhan-Ming Ying1, Zhan Wu1, Bin Tu1
1Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University , Changsha 410082, People's Republic of China.
Journal of the American Chemical Society
|July 18, 2017
Summary
Researchers developed a novel RNA sensor for fluorescently imaging microRNAs (miRs) in living tumor cells. This light-up RNA aptamer technology enables high-contrast imaging and quantitative analysis of miRs in real-time.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Light-up RNA aptamers are crucial for RNA imaging in living cells, aiding the study of RNA functions.
- Existing sensors lack the capability for microRNA (miR) imaging in mammalian cells.
- MicroRNAs play vital roles in gene regulation and disease, necessitating effective imaging tools.
Purpose of the Study:
- To develop a novel, genetically encoded RNA sensor for fluorescent imaging of microRNAs in living mammalian tumor cells.
- To create a light-up RNA aptamer-based sensor that responds to target miR binding with high-contrast fluorescence.
- To enable quantitative, ratiometric imaging of miRs in living cells through dual-emission capabilities.
Main Methods:
- Designed a light-up RNA aptamer sensor utilizing a sulforhodamine dye and a conjugated quencher.
- Employed a structural switching mechanism, similar to molecular beacons, for fluorescence activation upon miR binding.
- Integrated the RNA sensor into a tRNA scaffold for stable expression in tumor cells.
- Achieved dual-emission, ratiometric imaging by co-expressing the RNA sensor with Green Fluorescent Protein (GFP).
Main Results:
- Demonstrated a novel RNA sensor capable of high-contrast fluorescence imaging of target miRs in living tumor cells.
- Confirmed that the RNA sensor exhibits a light-up response upon hybridization with specific miRs.
- Successfully implemented ratiometric imaging, allowing for quantitative assessment of miR levels in living cells.
- Showcased the stable expression of the RNA sensor within a tRNA scaffold in tumor cells.
Conclusions:
- The developed RNA sensor represents a significant advancement for microRNA imaging in living cells.
- This novel sensor design offers a robust and sensitive platform for studying miR dynamics and functions.
- The approach provides a new paradigm for creating advanced light-up RNA sensors for diverse biological imaging applications.
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