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 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.

Keywords: binary deoxyribozyme; fluorescent sensors; mix-and-read probes; mutation analysis; ribosomal RNA.

FIGURE 1.

Principle of the BiDz assay. Two DNA strands, Dz_a and Dz_b, hybridize to the abutting positions of RNA analyte and form a catalytic core that cleaves a fluorophore- and a quencher-labeled reporter substrate. Fluorescence increases due to the spatial separation of the fluorophore from the quencher upon the substrate cleavage.

FIGURE 2.

Secondary structure model of E. coli 23S rRNA (Noller et al. 1981) with indicated fragments targeted by BiDz0 or BiDz-wt. (A) Domains I–III. (B) Domains IV–VI.

FIGURE 3.

Fluorescent response of the sensors BiDz0, BiDz-wt, or BiDz-m04 in the presence of different concentrations (0–800 pM) of in vitro 23S rRNA transcripts. (A) Wild-type RNA transcript (23S rRNA-wt) and (B) mutant m04 RNA transcript (23S rRNA-04). (Inset) Enlarged linear fragments of the curves in B. BiDz sensors were incubated with the transcripts for 1 h at 54°C.

FIGURE 4.

Selectivity of BiDz sensors. Fluorescence intensity of BiDz-wt(15) (dark gray bars) or BiDz-SNS(15) (light gray bars) in the presence of either synthetic oligonucleotide analytes (wt analyte or SNS analyte) (200 pM), 23S rRNA-wt (250 pM), wt E. coli cells or cells spiked with synthetic SNS analyte (200 pM). Blank sample contained no analyte. The results for three independent experiments were averaged; the errors are given as standard deviations.

FIGURE 5.

Quantification of 23S rRNA-04 in the mixtures of wt and m04 23S rRNA transcripts. (A) BiDz assay using either BiDz0 (open circles) or BiDz-m04 (filled circles) sensors. All samples contained 40 pM total 23S rRNA with different fractions of m04 RNA transcript. The values on y-axis represent fluorescence normalized by either the averaged fluorescence for all samples (for BiDz-wt) or maximum fluorescence (for BiDz-m04) achieved in the presence of 100% 23S rRNA-m04. (B) Primer extension assay. The samples contained the same fractions of m04 RNA transcript as in A.

FIGURE 6.

Normalized fluorescence intensity of BiDz0 (dark gray bars), BiDz-wt (striped bars), or BiDz-m04 (white bars) in the presence of nontransformed E. coli cells (6.25 × 10⁶ or 2.5 × 10⁷ cells/mL). For BiDz0 and BiDz-wt, the fluorescence values were normalized by the maximum fluorescence of the corresponding sensor achieved at saturation (in the presence of 2 × 10⁸ cells/mL). For BiDz-m04, the fluorescence values were normalized by the fluorescence of BiDz-wt at saturation.

FIGURE 7.

Level of the mutated 23S rRNA in E. coli cells transformed with pEC23M04 plasmid. (A) Normalized fluorescence intensity of either BiDz0 (open circles) or BiDz-m04 (filled circles) sensor in the presence of mixtures of E. coli cells containing a different amount of the cells transformed with pEC23M04 plasmid. The fluorescence values were normalized by either the averaged fluorescence for all samples (for BiDz-wt) or maximum fluorescence (for BiDz-m04) achieved in the presence of 100% transformed cell cultures. (B) Gel electrophoretic analysis of the products of primer extension assay. (TC) Transformed cells, (NC) nontransformed E. coli cells used as a control.