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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

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 semi- quantification 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.

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    Title
    • Expedited Quantification of Mutant Ribosomal RNA by Binary Deoxyribozyme (BiDz) Sensors
    Contributors
    Date Created
    2015-08-19
    Resource Type
  • Text
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    Identifier
    • Digital object identifier: 10.1261/rna.052613.115
    • Identifier Type
      International standard serial number
      Identifier Value
      1355-8382
    • Identifier Type
      International standard serial number
      Identifier Value
      1469-9001
    Note
    • The final version of this article, as published in RNA, can be viewed online at: http://rnajournal.cshlp.org/content/early/2015/08/19/rna.052613.115.full.pdf+html

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    This is a suggested citation. Consult the appropriate style guide for specific citation guidelines.

    Gerasimova, Y. V., Yakovchuk, P., Dedkova, L. M., Hecht, S. M., & Kolpashchikov, D. M. (2015). Expedited quantification of mutant ribosomal RNA by binary deoxyribozyme (BiDz) sensors. Rna, 21(10), 1834-1843. doi:10.1261/rna.052613.115

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