Intracellular Live-Cell RNA Imaging with Minimal Spectral Bleed-Through Using a Molecular Probe with Elongated Conjugation

High Resolution Image Download MS PowerPoint Slide Fluorescent RNA staining is an important tool in cellular imaging and biomedical research. However, developing RNA probes that are cell-permeable, minimize cross-photoexcitation, exhibit good RNA selectivity, and remain compatible with advanced imaging techniques continues to be challenging. Here, we report IND-BTZ, a deep-red RNA dye characterized by a significant Stokes shift (∼140 nm) and minimal spectral bleed-through. The probe performs well in live-cell imaging and targets mitochondrial RNA, as established by colocalization with MitoTracker Green and mitoGFP. However, in fixed cells, the probe demonstrates good RNA selectivity by staining nucleolar, nucleoplasmic, and cytoplasmic RNAs. Therefore, IND-BTZ represents an interesting dual-modal fluorescent probe that enables imaging without the need for poststaining wash steps. Notably, comparison with the commercially available SYTO green revealed approximately ∼50% signal leakage into the complementary TRITC channel. In contrast, IND-BTZ displayed significantly reduced leakage (∼7%) in the complementary FITC channel, indicating its compatibility with GFP-tagged proteins and multicolor imaging applications. Collectively, these features position IND-BTZ as an effective RNA imaging probe for biomedical research.