Switchable Fluorescent Light-Up Aptamers Based on Riboswitch Architectures

Tianhe Wang, Friedrich C. Simmel

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Fluorescent light-up RNA aptamers (FLAPs) such as Spinach or Mango can bind small fluorogens and activate their fluorescence. Here, we adopt a switching mechanism otherwise found in riboswitches and use it to engineer switchable FLAPs that can be activated or repressed by trigger oligonucleotides or small metabolites. The fluorophore binding pocket of the FLAPs comprises guanine (G) quadruplexes, whose critical nucleotides can be sequestered by corresponding anti-FLAP sequences, leading to an inactive conformation and thus preventing association with the fluorophore. We modified the FLAPs with designed toehold hairpins that carry either an anti-FLAP or an anti-anti-FLAP sequence within the loop region. The addition of an input RNA molecule triggers a toehold-mediated strand invasion process that refolds the FLAP into an active or inactive configuration. Several of our designs display close-to-zero leak signals and correspondingly high ON/OFF fluorescence ratios. We also modified purine aptamers to sequester a partial anti-FLAP or an anti-anti-FLAP sequence to control the formation of the fluorogen-binding conformation, resulting in FLAPs whose fluorescence is activated or deactivated in the presence of guanine or adenine. We demonstrate that switching modules can be easily combined to generate FLAPs whose fluorescence depends on several inputs with different types of input logic.

Original languageEnglish
Article numbere202302858
JournalAngewandte Chemie International Edition in English
Volume62
Issue number41
DOIs
StatePublished - 9 Oct 2023

Keywords

  • Fluorescent Light-up Aptamers
  • Purine Aptamer
  • Sensors
  • Toehold-Mediated Strand Displacement

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