Real-time mRNA measurement during an in vitro transcription and translation reaction using binary probes

Henrike Niederholtmeyer, Ling Xu, Sebastian J. Maerkl

Research output: Contribution to journalArticlepeer-review

30 Scopus citations

Abstract

In vitro transcription and translation reactions have become popular for a bottom-up approach to synthetic biology. Concentrations of the mRNA intermediate are rarely determined, although knowledge of synthesis and degradation rates could facilitate rational engineering of in vitro systems. We designed binary probes to measure mRNA dynamics during cell-free protein synthesis by fluorescence resonance energy transfer. We tested different mRNA variants and show that the location and sequence environment of the probe target sites are important parameters for probe association kinetics and output signal. Best suited for sensitive real-time quantitation of mRNA was a target site located in the 3′ untranslated region, which we designed to reduce secondary structure. We used this probe-target pair to refine our knowledge of mRNA dynamics in the commercially available PURE cell-free protein synthesis system and characterized the effect of TetR repressor on mRNA synthesis rates from a T7 promoter.

Original languageEnglish
Pages (from-to)411-417
Number of pages7
JournalACS Synthetic Biology
Volume2
Issue number8
DOIs
StatePublished - 16 Aug 2013
Externally publishedYes

Keywords

  • FRET
  • binary probes
  • cell-free protein synthesis
  • in vitro synthetic biology
  • mRNA dynamics
  • transcription

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