Engineering a Synthetic Dopamine-Responsive Riboswitch for in Vitro Biosensing

Svetlana V. Harbaugh*, Adam D. Silverman, Yaroslav G. Chushak, Kathryn Zimlich, Monica Wolfe, Walter Thavarajah, Michael C. Jewett, Julius B. Lucks, Jorge L. Chavez

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

The detection of chemicals using natural allosteric transcription factors is a powerful strategy for point-of-use molecular sensing, particularly using fieldable cell-free gene expression (CFE) systems. However, the reliance of detection schemes on characterized protein-based sensors limits the number of measurable analytes. One alternative solution to this issue is to develop new sensors by generating RNA aptamers against the target analyte and then incorporating them directly into a riboswitch scaffold for ligand-inducible genetic control of a reporter protein. However, this strategy has not generated more than a handful of successful portable cell-free molecular sensors. To address this gap, here we convert dopamine-binding aptamers into functional dopamine-sensing riboswitches that regulate gene expression in a freeze-dried CFE reaction. We then develop an assay for direct detection and semi-quantification of dopamine in human urine. We anticipate that this work will be broadly applicable for converting many in vitro-generated RNA aptamers into fieldable molecular diagnostics.

Original languageEnglish (US)
Pages (from-to)2275-2283
Number of pages9
JournalACS synthetic biology
Volume11
Issue number7
DOIs
StatePublished - Jul 15 2022

Keywords

  • TX-TL
  • aptamer
  • biosensing
  • cell-free gene expression
  • dopamine
  • human performance
  • riboswitch

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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