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

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.

Time course of large ribosomal subunit assembly in E. coli cells overexpressing a helicase inactive DbpA protein

“…1B), are large ribosome subunit precursors, the RNA composition of the ribosome profiles of cells overexpressing wild-type or R331A DbpA was investigated via a binary deoxyribozyme (biDz) sensor assay. This sensitive fluorescence assay has been used successfully in the past to detect bacterial rRNA both in vitro and in vivo (Gerasimova and Kolpashchikov 2013;Gerasimova et al 2015).…”

“…The sequences of the sensor strands and the fluorogenic reporter have been previously published (Gerasimova and Kolpashchikov 2013;Gerasimova et al 2015). For the assay, aliquots of the ribosome profile fractions were mixed with strands a and b of either of the sensors (15 nM each), and fluorogenic reporter substrate (200 nM) in a buffer containing 50 mM HEPES (pH 7.4), 50 mM MgCl 2 , 20 mM KCl, 120 mM NaCl, 0.03% Triton X-100 and 1% DMSO and heated at 95°C for 3 min.…”

Multiplex detection of extensively drug resistant tuberculosis using binary deoxyribozyme sensors

“…Additionally, a single BiDz sensor cleaves approximately 80 F-sub molecules per minute (16) resulting in a lower limit-of-detection compared to detection methods utilizing molecular beacon probes. Another important advantage of the BiDz approach is the ability to detect and differentiate both DNA and RNA analytes that differ by a single nucleotide (Gerasimova and Kolpashchikov 2010; Gerasimova et al 2015; Kolpashchikov 2007; Mokany et al 2010). To enable high selectivity of the sensors, the analyte binding arm of Dz b is designed short enough to enable formation of the catalytically active complex only in the presence of the fully matched analyte (Fig.…”

Interrogation of highly structured RNA with multicomponent deoxyribozyme probes at ambient temperatures

“…For example, such probes can be highly selective and, at the same time, able to tightly bind a specific nucleic acid target, which is generally unattainable with conventional hybridization probes (Demidov and Frank-Kamenetskii 2004). This property of MHP can be utilized to interrogate highly structured natural RNA targets (Nguyen et al 2011;Li et al 2012;Gentry et al 2015;Gerasimova et al 2015). For example, an MBP-based tricomponent probe was able to efficiently interrogate and, at the same time, discriminate single-nucleotide substitutions (SNSs) in a target containing a stem of up to 13 bp (Nguyen et al 2011).…”