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

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Current intracellular RNA fluorescence labeling often relies on MS2 labeling, which uses multiple protein labels attached to MS2 hairpin structures on the RNA of interest.
• These protein labels significantly increase the mass of the bound RNA, potentially affecting RNA biology and accessibility.
• Previous work demonstrated targeting of uridine-rich internal loops (URILs) with peptide nucleic acids (PNAs) with minimal structural perturbation.

Purpose of the Study:

• To develop and validate a new RNA and DNA tracking strategy that avoids protein fusion labels and minimizes structural alterations.
• To assess the efficacy of URIL-targeting fluorogenic bifacial PNAs (bPNAs) for intracellular RNA and RNP labeling in fixed and live cells.

Main Methods:

• Development of fluorogenic U-rich internal loop (FLURIL) tagging using bPNAs targeting URILs in RNA.
• Internal validation by comparing FLURIL tagging with MS2 labeling on the same RNA constructs.
• Direct comparison of FLURIL-tagged guide RNA (gRNA) with MS2-tagged gRNA for CRISPR-dCas9 labeled genomic loci in live U2OS cells.

Main Results:

• FLURIL probes successfully penetrated cell membranes and labeled RNAs and RNPs in both fixed and live cells.
• FLURIL tagging demonstrated superior signal-to-background ratios compared to MS2 labeling.
• FLURIL-tagged gRNA resulted in up to a 7-fold greater signal-to-background ratio than MS2-tagged gRNA for genomic loci labeling.

Conclusions:

• FLURIL tagging provides a versatile method for intracellular RNA and DNA tracking with a minimal molecular footprint.
• This technique is compatible with existing methods and offers improved signal detection over traditional protein-based labeling.
• FLURIL tagging represents a significant advancement for visualizing nucleic acids within living cells.