Live Cell Imaging of Single RNA Molecules with Fluorogenic Mango II Arrays

Overview

Journal Nat Commun

Specialty Biology

Date 2020 Mar 11

PMID 32152311

Citations 61

Authors

Adam D Cawte

Peter J Unrau

David S Rueda

Affiliations

Soon will be listed here.

Abstract

RNA molecules play vital roles in many cellular processes. Visualising their dynamics in live cells at single-molecule resolution is essential to elucidate their role in RNA metabolism. RNA aptamers, such as Spinach and Mango, have recently emerged as a powerful background-free technology for live-cell RNA imaging due to their fluorogenic properties upon ligand binding. Here, we report a novel array of Mango II aptamers for RNA imaging in live and fixed cells with high contrast and single-molecule sensitivity. Direct comparison of Mango II and MS2-tdMCP-mCherry dual-labelled mRNAs show marked improvements in signal to noise ratio using the fluorogenic Mango aptamers. Using both coding (β-actin mRNA) and long non-coding (NEAT1) RNAs, we show that the Mango array does not affect cellular localisation. Additionally, we can track single mRNAs for extended time periods, likely due to bleached fluorophore replacement. This property makes the arrays readily compatible with structured illumination super-resolution microscopy.

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References

Darzacq X, Shav-Tal Y, de Turris V, Brody Y, Shenoy S, Phair R . In vivo dynamics of RNA polymerase II transcription. Nat Struct Mol Biol. 2007; 14(9):796-806. PMC: 4942130. DOI: 10.1038/nsmb1280. View

Lionnet T, Czaplinski K, Darzacq X, Shav-Tal Y, Wells A, Chao J . A transgenic mouse for in vivo detection of endogenous labeled mRNA. Nat Methods. 2011; 8(2):165-70. PMC: 3076588. DOI: 10.1038/nmeth.1551. View

Hocine S, Raymond P, Zenklusen D, Chao J, Singer R . Single-molecule analysis of gene expression using two-color RNA labeling in live yeast. Nat Methods. 2012; 10(2):119-21. PMC: 3899799. DOI: 10.1038/nmeth.2305. View

Halstead J, Lionnet T, Wilbertz J, Wippich F, Ephrussi A, Singer R . Translation. An RNA biosensor for imaging the first round of translation from single cells to living animals. Science. 2015; 347(6228):1367-671. PMC: 4451088. DOI: 10.1126/science.aaa3380. View

Wang C, Han B, Zhou R, Zhuang X . Real-Time Imaging of Translation on Single mRNA Transcripts in Live Cells. Cell. 2016; 165(4):990-1001. PMC: 4905760. DOI: 10.1016/j.cell.2016.04.040. View

Morisaki T, Lyon K, DeLuca K, DeLuca J, English B, Zhang Z . Real-time quantification of single RNA translation dynamics in living cells. Science. 2016; 352(6292):1425-9. DOI: 10.1126/science.aaf0899. View

Yoon Y, Wu B, Buxbaum A, Das S, Tsai A, English B . Glutamate-induced RNA localization and translation in neurons. Proc Natl Acad Sci U S A. 2016; 113(44):E6877-E6886. PMC: 5098659. DOI: 10.1073/pnas.1614267113. View

Wu B, Eliscovich C, Yoon Y, Singer R . Translation dynamics of single mRNAs in live cells and neurons. Science. 2016; 352(6292):1430-5. PMC: 4939616. DOI: 10.1126/science.aaf1084. View

Bertrand E, Chartrand P, Schaefer M, Shenoy S, Singer R, Long R . Localization of ASH1 mRNA particles in living yeast. Mol Cell. 1998; 2(4):437-45. DOI: 10.1016/s1097-2765(00)80143-4. View

10.

Garcia J, Parker R . MS2 coat proteins bound to yeast mRNAs block 5' to 3' degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system. RNA. 2015; 21(8):1393-5. PMC: 4509929. DOI: 10.1261/rna.051797.115. View

11.

Garcia J, Parker R . Ubiquitous accumulation of 3' mRNA decay fragments in Saccharomyces cerevisiae mRNAs with chromosomally integrated MS2 arrays. RNA. 2016; 22(5):657-9. PMC: 4836640. DOI: 10.1261/rna.056325.116. View

12.

Tutucci E, Vera M, Biswas J, Garcia J, Parker R, Singer R . An improved MS2 system for accurate reporting of the mRNA life cycle. Nat Methods. 2017; 15(1):81-89. PMC: 5843578. DOI: 10.1038/nmeth.4502. View

13.

Wu B, Miskolci V, Sato H, Tutucci E, Kenworthy C, Donnelly S . Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences. Genes Dev. 2015; 29(8):876-86. PMC: 4403262. DOI: 10.1101/gad.259358.115. View

14.

Babendure J, Adams S, Tsien R . Aptamers switch on fluorescence of triphenylmethane dyes. J Am Chem Soc. 2003; 125(48):14716-7. DOI: 10.1021/ja037994o. View

15.

Paige J, Wu K, Jaffrey S . RNA mimics of green fluorescent protein. Science. 2011; 333(6042):642-6. PMC: 3314379. DOI: 10.1126/science.1207339. View

16.

Strack R, Disney M, Jaffrey S . A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA. Nat Methods. 2013; 10(12):1219-24. PMC: 3852148. DOI: 10.1038/nmeth.2701. View

17.

Filonov G, Moon J, Svensen N, Jaffrey S . Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution. J Am Chem Soc. 2014; 136(46):16299-308. PMC: 4244833. DOI: 10.1021/ja508478x. View

18.

Dolgosheina E, Jeng S, Panchapakesan S, Cojocaru R, Chen P, Wilson P . RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking. ACS Chem Biol. 2014; 9(10):2412-20. DOI: 10.1021/cb500499x. View

19.

Zhang J, Fei J, Leslie B, Han K, Kuhlman T, Ha T . Tandem Spinach Array for mRNA Imaging in Living Bacterial Cells. Sci Rep. 2015; 5:17295. PMC: 4661537. DOI: 10.1038/srep17295. View

20.

Sunbul M, Jaschke A . SRB-2: a promiscuous rainbow aptamer for live-cell RNA imaging. Nucleic Acids Res. 2018; 46(18):e110. PMC: 6182184. DOI: 10.1093/nar/gky543. View