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Related Concept Videos

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Related Experiment Video

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Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection
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LNA-enhanced DNA FIT-probes for multicolour RNA imaging.

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  • 1Department of Chemistry , Humboldt University Berlin , Brook-Taylor-Str. 2 , D-12489 Berlin , Germany .

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Researchers developed new red-emitting fluorescent probes for simultaneous RNA imaging. These quinoline blue (QB)-FIT probes enable sensitive detection of intracellular mRNA and multiplexed analysis, overcoming background autofluorescence.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Microscopy

Background:

  • Simultaneous imaging of intracellular RNA molecules in homogeneous solution presents challenges, requiring optimized, unambiguous staining methods.
  • Existing single dye forced intercalation (FIT) probes, using thiazole orange (TO) family dyes, primarily emit in the cyan and green spectrum.
  • Overcoming cellular autofluorescence and enabling multiplexed detection necessitates probes with emission in the red spectrum.

Purpose of the Study:

  • To develop novel chromophores for FIT probes emitting in the red spectrum (>600 nm) for enhanced RNA imaging.
  • To create a rapid screening method for identifying suitable thiazole orange (TO) family dyes for FIT probe applications.
  • To demonstrate the utility of new red-emitting probes for multiplexed RNA detection and intracellular mRNA imaging.

Main Methods:

  • Developed a submonomer approach for rapid analysis of various TO family dyes at different sequence positions.
  • Synthesized and characterized a novel carboxymethylated 4,4'-methine linked cyanine dye, named quinoline blue (QB), for FIT probes.
  • Employed wash-free fluorescent in situ hybridization and super-resolution microscopy (STED) for RNA localization in Drosophila melanogaster oocytes.

Main Results:

  • Identified quinoline blue (QB) as a novel chromophore for red-emitting FIT probes with a maximum emission at 605 nm.
  • QB-FIT probes exhibited a significant fluorescence enhancement of up to 195-fold upon binding to complementary RNA, exceeding previous base surrogates.
  • Demonstrated simultaneous detection of three different RNA targets using a mixture of BO-, TO-, and QB-FIT probes in homogeneous solution.
  • Successfully localized oskar mRNA and other polyadenylated mRNA in Drosophila melanogaster oocytes using QB- and TO-FIT probes.

Conclusions:

  • Novel red-emitting quinoline blue (QB)-FIT probes significantly enhance fluorescence upon RNA hybridization, enabling sensitive intracellular mRNA imaging.
  • The developed submonomer approach facilitates rapid discovery of new fluorescent dyes for molecular probes.
  • Multiplexed detection of RNA targets is achievable with a combination of FIT probes emitting across different spectral ranges, including red.
  • QB-FIT probes are valuable tools for high-resolution imaging of specific RNA molecules in biological samples, overcoming autofluorescence challenges.