Expedited quantification of mutant ribosomal RNA by binary deoxyribozyme (BiDz) sensors

Yulia V. Gerasimova, Petro Yakovchuk, Larisa Dedkova, Sidney Hecht, Dmitry M. Kolpashchikov

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

Mutations in ribosomal RNA (rRNA) have traditionally been detected by the primer extension assay, which is a tedious and multistage procedure. Here, we describe a simple and straightforward fluorescence assay based on binary deoxyribozyme (BiDz) sensors. The assay uses two short DNA oligonucleotides that hybridize specifically to adjacent fragments of rRNA, one of which contains a mutation site. This hybridization results in the formation of a deoxyribozyme catalytic core that produces the fluorescent signal and amplifies it due to multiple rounds of catalytic action. This assay enables us to expedite semiquantification of mutant rRNA content in cell cultures starting from whole cells, which provides information useful for optimization of culture preparation prior to ribosome isolation. The method requires less than a microliter of a standard Escherichia coli cell culture and decreases analysis time from several days (for primer extension assay) to 1.5 h with hands-on time of ∼10 min. It is sensitive to single-nucleotide mutations. The new assay simplifies the preliminary analysis of RNA samples and cells in molecular biology and cloning experiments and is promising in other applications where fast detection/ quantification of specific RNA is required.

Original languageEnglish (US)
Pages (from-to)1834-1843
Number of pages10
JournalRNA
Volume21
Issue number10
DOIs
StatePublished - Oct 1 2015

Keywords

  • Binary deoxyribozyme
  • Fluorescent sensors
  • Mix-and-read probes
  • Mutation analysis
  • Ribosomal RNA

ASJC Scopus subject areas

  • Molecular Biology

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