Fluorogenic U-rich internal loop (FLURIL) tagging with bPNA enables intracellular RNA and DNA tracking

Abstract

We introduce herein a new strategy for intracellular RNA and DNA tracking that is robust, orthogonal and complementary to existing methods: Fluorogenic U-Rich Internal Loop (FLURIL) tagging with cell-permeable fluorophore-labeled bifacial Peptide Nucleic Acids (fbPNAs). Our approach uses an 8-nt (U4xU4) U-rich internal loop (URIL) in the RNA of interest (ROI) as a compact labeling site for fluorogenic triplex hybridization with a bPNA probe (~1 kD). FLURIL tagging thus replaces a 4 bp duplex stem with a labeled 4-base-triple hybrid stem of similar structure and mass. In contrast to existing strategies for RNA tracking, FLURIL tagging can be applied to internal, genetically encoded URIL RNA sites with minimal structural perturbation, co-expression of protein-fusion labels or significant increase in molecular weight and steric bulk. We demonstrate effective FLURIL tagging of intracellular (HEK-293) RNAs, ribonucleoprotein (RNP) complexes and live cell (U2-OS) tracking of genomic loci. FLURIL tracking was internally validated by direct comparison with the most widely used live-cell RNA labeling method, MS2-labeling with MCP-HaloTag and Janelia Fluor dyes. In addition, FLURIL-tagging correctly reported on the endogenous RNP in HEK293 cells formed from TAR DNA binding protein 43 (TDP-43-tdTomato) and UG repeat RNA. The FLURIL strategy was also successfully applied to guide RNA (gRNA) in CRISPR-dCas complexes to enable live cell tracking of a low-copy number genomic locus (IDR3), internally benchmarked against MS2/HaloTag labeling of CRISPR-Sirius gRNA targeted to a proximal locus (IDR2). Notably, FLURIL-tagged IDR2 exhibited similar brightness as loci targeted by CRISPR-Sirius gRNA complexes, which bear 8-MS2 hairpins for protein labeling. Together, these experiments show that FLURIL tagging can simply and reliably track intracellular RNA, RNPs, and DNA, with a streamlined molecular footprint relative to other methods. Importantly, these data also indicate that FLURIL tagging is fully compatible with existing labeling methods without crosstalk and may be used to broaden the scope of intracellular RNA and DNA tracking. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/501035v1_figS1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1e6cc24org.highwire.dtl.DTLVardef@1646707org.highwire.dtl.DTLVardef@a80268org.highwire.dtl.DTLVardef@363193_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOScheme 1.C_FLOATNO FLURIL-tagging of RNAs with bPNA probes. (a) Triplex hybridization of a U-rich internal loop (URIL) with bPNA (blue) via base triple formation between the melamine base (M) and two uracil bases (inset). (b) General schematic of labeling strategy described herein. An RNA of interest is engineered to contain an URIL and expressed within the cell, with a fluorogenic bPNA probe introduced via cell culture media. Successful URIL targeting is reported by an increase in emission (green) and confirmed by a previously established RNA binding protein with a fluorescent protein (red) fusion. C_FIG