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Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers
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Programmable RNA detection with a fluorescent RNA aptamer using optimized three-way junction formation.

Yuichi Furuhata1, Mizuki Kobayashi2, Ryo Maruyama3

  • 1Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566, Japan.

RNA (New York, N.Y.)
|February 13, 2019
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Summary

Researchers developed novel RNA detection probes using Broccoli and Baby Spinach aptamers. These probes offer sensitive, programmable detection of target RNAs with significant fluorescence enhancement, aiding cellular function studies and disease diagnostics.

Keywords:
Baby SpinachBroccoliRNA sensorfluorescent RNAthree-way junction

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Biotechnology

Background:

  • Endogenous RNA detection is crucial for understanding cellular processes and diagnosing diseases.
  • Developing cost-effective and user-friendly RNA detection probes is a significant goal.
  • Fluorescent RNA aptamers, like Spinach and its variants, offer a promising avenue for RNA sensing.

Purpose of the Study:

  • To characterize the higher-order structure of the Broccoli RNA aptamer.
  • To design and develop novel structure-switching aptamer probes for programmable RNA detection.
  • To achieve highly sensitive and specific detection of target RNAs using engineered aptamer probes.

Main Methods:

  • Experimental characterization of the Broccoli RNA aptamer structure.
  • Design of Broccoli-based RNA detection probes (BRD probes) incorporating complementary sequences.
  • Construction of a three-way junction (MT2) for enhanced aptamer refolding and signal generation.
  • Application of the MT2 junction strategy to create Baby Spinach-based RNA detection probes (BSRD probes).

Main Results:

  • The Broccoli aptamer's G-quadruplex-based DFHBI-1T recognition region was experimentally characterized.
  • BRD and BSRD probes demonstrated significant fluorescence enhancements (up to 48- and 140-fold, respectively) upon target RNA binding.
  • The probes achieved sensitive detection of target RNAs down to 160 nM (BRD) and 5 nM (BSRD).
  • The MT2 three-way junction facilitated efficient aptamer refolding and programmable RNA detection.

Conclusions:

  • The study successfully characterized the Broccoli aptamer structure and developed highly sensitive, programmable RNA detection probes.
  • The engineered MT2 three-way junction is a versatile platform for structure-switching aptamer probe design.
  • These novel probes hold potential for advancing RNA sensing applications in research and diagnostics.