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Related Experiment Video

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A Fluorescent Split Aptamer for Visualizing RNA-RNA Assembly In Vivo.

Khalid K Alam1,2, Kwaku D Tawiah1,2, Matthew F Lichte1,2

  • 1Department of Biochemistry, University of Missouri , Columbia, Missouri 65211, United States.

ACS Synthetic Biology
|May 27, 2017
PubMed
Summary

Researchers developed the Split-Broccoli system, the first functional split-aptamer system operating in living cells. This genetically encoded tool monitors RNA-RNA hybridization events non-destructively, advancing synthetic biology and genetic circuit engineering.

Keywords:
RNA AND gateRNA interactionRNA synthetic biologybinary aptamercotranscriptional RNA assemblyfluorescence complementation

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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Biochemistry

Background:

  • RNA-RNA assembly is crucial for biological processes and synthetic genetic circuits.
  • Existing methods for monitoring RNA assembly in cells, like fluorescent reporters, have limitations.
  • RNA aptamers offer a more direct way to report RNA-level events by sequestering dyes.

Purpose of the Study:

  • To engineer a split-aptamer system that functions in vivo for monitoring RNA-RNA hybridization.
  • To leverage the properties of the Broccoli aptamer for in vivo split-aptamer system construction.
  • To create a genetically encoded, nondestructive platform for studying RNA-RNA interactions.

Main Methods:

  • Designed the Split-Broccoli system using a thermostable, three-way junction RNA architecture.
  • Investigated the kinetics of Split-Broccoli assembly in vitro and in vivo.
  • Fused the Split-Broccoli system to an RNA Toehold switch to demonstrate functional modularity.

Main Results:

  • The Split-Broccoli system activates fluorescence upon hybridization of two RNA strands.
  • Functional assembly kinetics followed second-order dynamics in vitro and improved with co-transcription.
  • Demonstrated digital in vivo fluorescence and functional modularity when integrated with an RNA Toehold switch.

Conclusions:

  • Split-Broccoli is the first functional split-aptamer system demonstrated to operate in vivo.
  • This system provides a genetically encoded and nondestructive method to monitor and engineer RNA-RNA hybridization.
  • Split-Broccoli can serve as an all-RNA AND gate or a tool for monitoring RNA-RNA hybrid assembly in biological systems.