ArticleBio-protocol2021
A Multi-color Bicistronic Biosensor to Compare the Translation Dynamics of Different Open Reading Frames at Single-molecule Resolution in Live Cells.
Article in Bio-protocol, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 3 papers.
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Who cites it
3 citing papers in PubMed, 4 citations in OpenAlex.
- Imaging and Quantifying Ribosomal Frameshifting Dynamics with Single-RNA Precision in Live Cells.Methods in molecular biology (Clifton, N.J.) · 2025Article
- Development of label-free light-controlled gene expression technologies using mid-IR and terahertz light.Frontiers in bioengineering and biotechnology · 2024Review
- Visualization, Quantification, and Modeling of Endogenous RNA Polymerase II Phosphorylation at a Single-copy Gene in Living Cells.Bio-protocol · 2022Article
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
Abstract
Here, we describe how to image and quantitate the translation dynamics of a bicistronic biosensor that we recently created to fairly compare cap-dependent and IRES-mediated translation at single-molecule resolution in live human cells. This technique employs a pair of complementary intrabodies loaded into living cells that co-translationally bind complementary epitopes in the two separate ORFs of the bicistronic biosensor. This causes the biosensor to fluoresce in different colors depending on which ORF/epitopes are translated. Using the biosensor together with high-resolution fluorescence microscopy and single-molecule tracking analysis allows for the quantitative comparison of translation dynamics between the two ORFs at a resolution of tens-of-nanometers in space and sub-seconds in time, which is not possible with more traditional GFP or luciferase reporters. Since both ORFs are on the same biosensor, they experience the same microenvironment, allowing a fair comparison of their relative translational activities. In this protocol, we describe how to get this assay up and running in cultured human cells so that translation dynamics can be studied under both normal and stressful cellular conditions. We also provide a number of useful tips and notes to help express components at appropriate levels inside cells for optimal live cell imaging. Graphical abstract: Steps required for 3-color single-molecule translation imaging and analysis.
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Registered trials
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