Split Spinach Aptamer for Highly Selective Recognition of DNA and RNA at Ambient Temperatures

Abstract

Split spinach aptamer (SSA) probes for fluorescent analysis of nucleic acids were designed and tested. In SSA design, two RNA or RNA/DNA strands hybridized to a specific nucleic acid analyte and formed a binding site for low-fluorescent 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) dye, which resulted in up to a 270-fold increase in fluorescence. The major advantage of the SSA over state-of-the art fluorescent probes is high selectivity: it produces only background fluorescence in the presence of a single-base-mismatched analyte, even at room temperature. SSA is therefore a promising tool for label-free analysis of nucleic acids at ambient temperatures.

Keywords: RNA; aptameric sensor; high selectivity; label-free probes; split probes.

Figure 1

General design and fluorescent response of the split spinach aptamer (SSA) probe and the fluorescent reposed of SSA^r and SSA^d probes. A) Two strands, SSA_m and SSA_f hybridize to a specific DNA or RNA analyte and re-form a binding site for DFHBI organic dye. Binding of the dye by the aptamer results in fluorescent increase. Dashed lines represent analyte-binding arms, which were DNA in SSA^d or RNA in SSA^r. Dotted line is either diuridylate (UU) linker for SSA^r or triethylene glycol for SSA^d (see Fig. S1 for detailed design). B) Fluorescent response of SSA^r_m (2.6 µM), SSA^r_f (3.6 µM) and DFHBI dye (1 µM) in the absence or presence of fully matched analyte A_m^r (1.38 µM). Emission spectrum (λ_ex = 450 nm) were recorded after 90 min of incubation. C) Fluorescent response of SSA^d_m (2 µM), SSA^d_f (3.6 µM) and DFHBI dye (2 µM) in the absence or presence of fully matched DNA analyte A_m^d (5.5 µM). Emission spectrum (λ_ex = 450 nm) were recorded after 30 min of incubation.

Figure 2

Kinetics and limit of detection (LOD) of the SSA probes. A) Time dependence of fluorescent response of SSA^r and RNA analytes (left) and SSA^d and DNA analytes (right). The apparent difference in signal-to-noise ratio in comparison with Figure 1 B and C is due to the different analyte concentrations. Reaction mixtures contained: 1 µM DFHBI, 2.6 µM SSA^r_m and 3.6 µM SSA^r_f, 275 nM RNA analytes; or 2 µM DFHBI, 2 µM SSA^d_m and 3.6 µM SSA^d_f, 275 nM DNA analytes. B) Limits of detection (LOD) for SSA^r and SSA^d after 30 min of incubation. Averaged data from three independent experiments with standard deviations are presented.

Figure 3

Selectivity of the SSA probes. A) Fluorescence response of SSA_r in the presence of 100 nM of either matched (A^r_m) or single base mismatched (A^r_mm) RNA analytes. B) Fluorescence response of SSA^d in the presence of 100 nM of either matched (A^d_m) or single base mismatched (A^d_mm) DNA analytes. The data are average values of 3 independent experiments with standard deviations. C) Photograph of the SSA^d samples from panel B upon excitation with transilluminator. The controls samples were as follows: DFHBI, DFHBI dye only; SAA, DFHBI dye, SSA_m, and SSA_f (no analyte). The concentrations were as specified in Figure 2 legend.