Single enzyme RT-PCR of full-length ribosomal RNA

Michael J. Hammerling, Danielle J. Yoesep, Michael C. Jewett*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

The ribosome is a two-subunit, macromolecular machine composed of RNA and proteins that carries out the polymerization of a-amino acids into polypeptides. Efforts to engineer ribosomal RNA (rRNA) deepen our understanding of molecular translation and provide opportunities to expand the chemistry of life by creating ribosomes with altered properties. Toward these efforts, reverse transcription PCR (RT-PCR) of the entire 16S and 23S rRNAs, which make up the 30S small subunit and 50S large subunit, respectively, is important for isolating desired phenotypes. However, reverse transcription of rRNA is challenging due to extensive secondary structure and post-transcriptional modifications. One key challenge is that existing commercial kits for RT-PCR rely on reverse transcriptases that lack the extreme thermostability and processivity found in many commercial DNA polymerases, which can result in subpar performance on challenging templates. Here, we develop methods employing a synthetic thermostable reverse transcriptase (RTX) to enable and optimize RT-PCR of the complete Escherichia coli 16S and 23S rRNAs. We also characterize the error rate of RTX when traversing the various posttranscriptional modifications of the 23S rRNA. We anticipate that this work will facilitate efforts to study and characterize many naturally occurring long RNAs and to engineer the translation apparatus for synthetic biology.

Original languageEnglish (US)
Article numberysaa028
JournalSynthetic Biology
Volume5
Issue number1
DOIs
StatePublished - 2021

Keywords

  • Directed evolution
  • In vitro
  • RT-PCR
  • Ribosome construction
  • Synthetic biology

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomaterials
  • Biomedical Engineering
  • Agricultural and Biological Sciences (miscellaneous)

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