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The structure and stability of mRNA molecules regulates gene expression, as mRNAs are a key step in the pathway from gene to protein. In eukaryotes, the half-life of mRNA varies from a few minutes up to several days. mRNA stability is essential in growth and development. The absence of the proteins regulating its stability, such as tristetraprolin in mice, can cause systemic issues, including bone marrow overgrowth, inflammation, and autoimmunity.
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Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons
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Quantitative Detection and Real-Time Monitoring of Endogenous mRNA at the Single Live Cell Level Using a Ratiometric

Xufei Feng1, Wenjie Kang1, Xuping Wu2

  • 1Joint International Research Laboratory of Animal Health and Food Safety & Single Molecule Nanometry Laboratory (Sinmolab) , Nanjing Agricultural University , Nanjing 210095 , China.

ACS Applied Materials & Interfaces
|July 23, 2019
PubMed
Summary

Researchers developed a novel ratiometric molecular beacon (RMB) for real-time, quantitative detection of messenger ribonucleic acid (mRNA) in live cells. This advanced probe offers high sensitivity and specificity, overcoming limitations of traditional methods for endogenous mRNA analysis.

Keywords:
Hsp27 mRNAendogenous mRNA subcellular localizationfluorescence signal co-localization analysislive cell detectionquantitative endogenous mRNA measurementsratiometric molecular beacon (RMB)real-time endogenous mRNA monitoring

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cell Biology

Background:

  • Traditional messenger ribonucleic acid (mRNA) detection methods require sample purification or cell fixation, hindering real-time analysis in live cells.
  • Accurate quantification of endogenous mRNA within single live cells is crucial for understanding cellular processes.
  • Existing techniques face challenges with specificity, sensitivity, and susceptibility to nuclease degradation.

Purpose of the Study:

  • To develop a novel ratiometric molecular beacon (RMB) for real-time, quantitative detection of endogenous mRNA in single live cells.
  • To overcome the limitations of traditional mRNA detection methods, including the need for purification and the inability to monitor live cells.
  • To establish a robust tool for analyzing mRNA dynamics, localization, and copy numbers within individual living cells.

Main Methods:

  • Design and synthesis of a single-strand stem-loop structured ratiometric molecular beacon (RMB) with phosphorothioate modifications and 2'-O-methyl RNA backbone.
  • Incorporation of a reporter dye, quencher, and reference dye within the RMB for ratiometric fluorescence measurement.
  • Hybridization-based mechanism where target mRNA binding opens the stem-loop, separating reporter and quencher, enabling quantitative detection via fluorescence ratio.
  • In vitro and in vivo validation of RMB performance, including specificity, sensitivity, antidegradation capability, and application in live cell imaging.

Main Results:

  • The designed RMB demonstrated high signal-to-noise ratio, sensitivity, specificity, and antidegradation capability for detecting Hsp27 mRNA in vitro and in live cells.
  • The ratiometric detection approach, coupled with reference dye and co-localization analysis, corrected for variations in probe delivery and nuclease degradation.
  • Successful application of RMB for subcellular localization, quantitative copy number measurements, and real-time monitoring of endogenous mRNA in live cells.
  • Phosphorothioate and 2'-O-methyl RNA modifications effectively reduced non-specific opening of the molecular beacon.

Conclusions:

  • The developed ratiometric molecular beacon (RMB) provides a powerful tool for real-time, quantitative endogenous mRNA detection at the single live cell level.
  • This method overcomes critical limitations of traditional techniques, enabling precise analysis of mRNA dynamics in living systems.
  • The RMB technology holds significant potential for advancing research in molecular and cell biology, diagnostics, and drug discovery.