ArticleBiophysical journal2012
Fluorescence fluctuation spectroscopy enables quantitative imaging of single mRNAs in living cells.
Article in Biophysical journal, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 134 papers.
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Who cites it
134 citing papers in PubMed, 214 citations in OpenAlex.
- Single-cell transcriptional dynamics in a living vertebrate.Cell systems · 2026Article
- Synaptic Proteostasis in Health and Disease.Journal of neurochemistry · 2026Review
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- Establishing MS2-MCP-based single-molecule RNA visualization in Schizosaccharomyces pombe.Journal of cell science · 2026Article
- A series of spontaneously blinking dyes for super-resolution microscopy.Nature methods · 2026Article
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- Paraspeckle condensation is controlled via TDP-43 polymerization and linked to neuroprotection.Nature cell biology · 2026Article
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- Establishing MS2-MCP-based single-molecule RNA visualization inbioRxiv : the preprint server for biology · 2026Article
- Lighting Up Endogenous RNA: Fluorescent Aptamer Sensors for Live-Cell Imaging.Chemical & biomedical imaging · 2026Review
- Amplified MS2 labeling reveals heterogeneous dynamics of RNA granules in the live murine brain.bioRxiv : the preprint server for biology · 2026Article
- RNA-stabilized coat proteins for sensitive and simultaneous imaging of distinct single mRNAs in live cells.Nature methods · 2026Article
- Orthogonal RNA-regulated destabilization domains for three-color RNA imaging with minimal RNA perturbation.Nature methods · 2026Article
- Imaging Spatiotemporal RNA and Protein Dynamics in Mouse Embryos.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Single-molecule live-cell RNA imaging with CRISPR-Csm.Nature biotechnology · 2025Article
- Visualizing genetic information flow in space and time.Science China. Life sciences · 2025Review
- A non-coding role for trypanosome VSG transcripts in allelic exclusion.Nucleic acids research · 2025Article
- Single-protein/RNA imaging reveals ZNF598 as a limiting factor in resolving collided ribosomes.The EMBO journal · 2025Article
- FGF and BMP pathway genes are expressed in segmentally iterated domains during segmentation of germ layers in a tardigrade.bioRxiv : the preprint server for biology · 2025Article
- Optogenetic storage and release of protein and mRNA in live cells and animals.Nature communications · 2025Article
74 more citing papers are in PubMed but not listed here.
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
Abstract
Imaging mRNA with single-molecule sensitivity in live cells has become an indispensable tool for quantitatively studying RNA biology. The MS2 system has been extensively used due to its unique simplicity and sensitivity. However, the levels of the coat protein needed for consistent labeling of mRNAs limits the sensitivity and quantitation of this technology. Here, we applied fluorescence fluctuation spectroscopy to quantitatively characterize and enhance the MS2 system. Surprisingly, we found that a high fluorescence background resulted from inefficient dimerization of fluorescent protein (FP)-labeled MS2 coat protein (MCP). To mitigate this problem, we used a single-chain tandem dimer of MCP (tdMCP) that significantly increased the uniformity and sensitivity of mRNA labeling. Furthermore, we characterized the PP7 coat protein and the binding to its respective RNA stem loop. We conclude that the PP7 system performs better for RNA labeling. Finally, we used these improvements to study endogenous β-actin mRNA, which has 24xMS2 binding sites inserted into the 3' untranslated region. The tdMCP-FP allowed uniform RNA labeling and provided quantitative measurements of endogenous mRNA concentration and diffusion. This work provides a foundation for quantitative spectroscopy and imaging of single mRNAs directly in live cells.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.