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

Yufeng Liang, Sydney Willey, Yu-Chieh Chung, Yi-Meng Lo, Shiqin Miao, Sarah Rundell, Li-Chun Tu () and Dennis Bong ()
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Yufeng Liang: The Ohio State University
Sydney Willey: The Ohio State University
Yu-Chieh Chung: The Ohio State University
Yi-Meng Lo: The Ohio State University
Shiqin Miao: The Ohio State University
Sarah Rundell: The Ohio State University
Li-Chun Tu: The Ohio State University
Dennis Bong: The Ohio State University

Nature Communications, 2023, vol. 14, issue 1, 1-12

Abstract: 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.

Date: 2023
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DOI: 10.1038/s41467-023-38579-2

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