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Riboswitches

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Riboswitches are non-coding mRNA domains that regulate the transcription and translation of downstream genes without the help of proteins. Riboswitches bind directly to a metabolite and can form unique stem-loop or hairpin structures in response to the amount of the metabolite present. They have two distinct regions – a metabolite-binding aptamer and an expression platform.
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Riboswitches are RNA elements that regulate gene expression by altering their secondary structures in response to specific effector molecules. These elements, located in the leader regions of certain mRNAs, act as transcriptional regulators by toggling between alternative conformations to control downstream gene expression. Riboswitch-mediated regulation is a precise mechanism for modulating biosynthetic pathways, as exemplified by the riboflavin biosynthesis pathway in Bacillus...
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Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers
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Switchable Fluorescent Light-Up Aptamers Based on Riboswitch Architectures.

Tianhe Wang1, Friedrich C Simmel1

  • 1Physics of Synthetic Biological Systems-E14, Department of Bioscience, TUM School of Natural Science, Technische Universität München, Am Coulombwall 4a, 85748, Garching, Germany.

Angewandte Chemie (International Ed. in English)
|May 10, 2023
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Summary

Researchers engineered switchable fluorescent light-up RNA aptamers (FLAPs) that can be controlled by RNA or small molecules. These novel FLAPs offer high ON/OFF ratios and enable complex logic-based molecular sensing applications.

Keywords:
Fluorescent Light-up AptamersPurine AptamerSensorsToehold-Mediated Strand Displacement

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Synthetic Biology

Background:

  • Fluorescent light-up RNA aptamers (FLAPs) like Spinach and Mango bind fluorogens to emit fluorescence.
  • Existing FLAPs lack dynamic control over their fluorescence output.
  • Riboswitches utilize switching mechanisms for gene regulation.

Purpose of the Study:

  • To engineer switchable FLAPs controllable by external inputs.
  • To develop FLAPs with tunable activation and repression mechanisms.
  • To create RNA-based sensors with high signal-to-noise ratios.

Main Methods:

  • Engineered FLAPs with toehold hairpins containing anti-FLAP or anti-anti-FLAP sequences.
  • Utilized toehold-mediated strand invasion for conformational switching.
  • Modified purine aptamers to control fluorogen-binding pocket formation.
  • Integrated switching modules for multi-input logic gates.

Main Results:

  • Developed switchable FLAPs activated/repressed by trigger oligonucleotides or metabolites.
  • Achieved near-zero leak signals and high ON/OFF fluorescence ratios.
  • Demonstrated metabolite-dependent FLAP control (guanine, adenine).
  • Successfully created FLAPs responding to multiple inputs with diverse logic.

Conclusions:

  • Switchable FLAPs offer a versatile platform for RNA-based sensing and computation.
  • The engineered systems provide precise control over fluorescence output.
  • This work expands the toolkit for synthetic biology and molecular diagnostics.