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Updated: Aug 18, 2025
Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons
Published on: August 6, 2014
Hierarchical DNA branch assembly-encoded fluorescent nanoladders for single-cell transcripts imaging
Xiaowen Cao1, Feng Chen1, Jing Xue1
1Institute of Analytical Chemistry and Instrument for Life Science, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xianning West Road, Xi'an, Shaanxi 710049, P.R. China.
Researchers developed a new method for visualizing single RNA molecules within cells. This technique uses DNA nanoladders to amplify signals, enabling highly multiplexed and accurate transcript imaging with reduced background noise.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Single-cell transcriptomics is crucial for understanding cellular function.
- Current methods for spatial transcript imaging face limitations in signal specificity and multiplexing capabilities.
Purpose of the Study:
- To develop a novel method for denoised and highly multiplexed single-molecule transcript imaging.
- To overcome the limitations of existing techniques in spatial visualization of RNA.
Main Methods:
- Hierarchical DNA branch assembly-encoded fluorescent nanoladders were engineered.
- RNA-primed rolling circle amplification was employed, utilizing circular DNAzymes to prevent nonspecific amplification.
- Programmable DNA branch assembly was used to encode virtual signals for Fluorescence In Situ Hybridization (FISH).
Main Results:
- The method achieved significant reduction in nonspecific amplification in fixed cells (from 16 to nearly zero nonspecific spots per cell).
- Simultaneous quantitation of nine transcripts was achieved using only two detection spectral channels.
- Accurate RNA profiling and diverse transcript localization patterns were demonstrated in cancer cells.
Conclusions:
- The developed nanoladder system provides denoised and highly multiplexed signal amplification for single-molecule transcript imaging.
- This technique enables precise spatial profiling of RNA in various cell types, offering insights into spatial gene regulation.
- The method holds potential for advancing single-cell analysis and understanding complex biological processes.
