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Intracellular RNA and DNA tracking by uridine-rich internal loop tagging with fluorogenic bPNA

Author

Listed:
  • Yufeng Liang

    (The Ohio State University
    The Ohio State University)

  • Sydney Willey

    (The Ohio State University
    The Ohio State University
    The Ohio State University)

  • Yu-Chieh Chung

    (The Ohio State University)

  • Yi-Meng Lo

    (The Ohio State University
    The Ohio State University)

  • Shiqin Miao

    (The Ohio State University
    The Ohio State University)

  • Sarah Rundell

    (The Ohio State University
    The Ohio State University)

  • Li-Chun Tu

    (The Ohio State University
    The Ohio State University
    The Ohio State University)

  • Dennis Bong

    (The Ohio State University
    The Ohio State University)

Abstract

The most widely used method for intracellular RNA fluorescence labeling is MS2 labeling, which generally relies on the use of multiple protein labels targeted to multiple RNA (MS2) hairpin structures installed on the RNA of interest (ROI). While effective and conveniently applied in cell biology labs, the protein labels add significant mass to the bound RNA, which potentially impacts steric accessibility and native RNA biology. We have previously demonstrated that internal, genetically encoded, uridine-rich internal loops (URILs) comprised of four contiguous UU pairs (8 nt) in RNA may be targeted with minimal structural perturbation by triplex hybridization with 1 kD bifacial peptide nucleic acids (bPNAs). A URIL-targeting strategy for RNA and DNA tracking would avoid the use of cumbersome protein fusion labels and minimize structural alterations to the RNA of interest. Here we show that URIL-targeting fluorogenic bPNA probes in cell media can penetrate cell membranes and effectively label RNAs and RNPs in fixed and live cells. This method, which we call fluorogenic U-rich internal loop (FLURIL) tagging, was internally validated through the use of RNAs bearing both URIL and MS2 labeling sites. Notably, a direct comparison of CRISPR-dCas labeled genomic loci in live U2OS cells revealed that FLURIL-tagged gRNA yielded loci with signal to background up to 7X greater than loci targeted by guide RNA modified with an array of eight MS2 hairpins. Together, these data show that FLURIL tagging provides a versatile scope of intracellular RNA and DNA tracking while maintaining a light molecular footprint and compatibility with existing methods.

Suggested Citation

  • Yufeng Liang & Sydney Willey & Yu-Chieh Chung & Yi-Meng Lo & Shiqin Miao & Sarah Rundell & Li-Chun Tu & Dennis Bong, 2023. "Intracellular RNA and DNA tracking by uridine-rich internal loop tagging with fluorogenic bPNA," Nature Communications, Nature, vol. 14(1), pages 1-12, December.
  • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-38579-2
    DOI: 10.1038/s41467-023-38579-2
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    References listed on IDEAS

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    1. X. Rosa Ma & Mercedes Prudencio & Yuka Koike & Sarat C. Vatsavayai & Garam Kim & Fred Harbinski & Adam Briner & Caitlin M. Rodriguez & Caiwei Guo & Tetsuya Akiyama & H. Broder Schmidt & Beryl B. Cummi, 2022. "TDP-43 represses cryptic exon inclusion in the FTD–ALS gene UNC13A," Nature, Nature, vol. 603(7899), pages 124-130, March.
    2. Anna-Leigh Brown & Oscar G. Wilkins & Matthew J. Keuss & Sarah E. Hill & Matteo Zanovello & Weaverly Colleen Lee & Alexander Bampton & Flora C. Y. Lee & Laura Masino & Yue A. Qi & Sam Bryce-Smith & Ar, 2022. "TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A," Nature, Nature, vol. 603(7899), pages 131-137, March.
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