Fluorescence detection of single-nucleotide polymorphisms with a single, self-complementary, triple-stem DNA probe

Abstract

Singled out for its singularity: In a single-step, single-component, fluorescence-based method for the detection of single-nucleotide polymorphisms at room temperature, the sensor is comprised of a single, self-complementary DNA strand that forms a triple-stem structure. The large conformational change that occurs upon binding to perfectly matched (PM) targets results in a significant increase in fluorescence (see picture; F = fluorophore, Q = quencher).

Figure 1

(A) Mechanism of the SNP sensor. In the presence of a perfectly-matched (PM) target, the folded triple-stem DNA structure is disrupted and fluoresces. In contrast, single-base mismatched (1MM) or two-base mismatched (2MM) targets do not destabilize the discontinuous duplex, and the probe maintains its triple-stem structure with quenched fluorescence. (B) Emission spectra of the triple-stem probe (1) (0.5 μM) following incubation at room temperature with PM target (2), 1MM target (3), 2MM target (4), or no target. The fluorescence signal was obtained at λ_ex= 590 nm and λ_em= 610 nm, and all targets were 17 bases in length.

Figure 2

The triple-stem probe (1) displays excellent discrimination against mismatches. (A) Proposed phase transitions of the triple-stem probe in the presence of targets at different temperatures. (B) The SNP sensor retains its discrimination functionality up to 60 °C. Thermal denaturation curves of the probe only, or hybridized with PM target (2), 1MM target (3), or 2MM target (4). (C) Gel image of the triple-stem probe only (lanes 1 and 4), PM target (2)-probe samples (lanes 2 and 5) and 1MM target (3)-probe samples (lanes 3 and 6). One set of samples was equilibrated for three hours (lanes, 1-3), the other was equilibrated for three days (lanes, 4-6). (D) A calibration curve of PM target (2) and 1MM target (3) for the triple-stem probe. The inset shows the concentration-dependence of discrimination factor of 17-base targets in the presence of 0.5 μM of the probe.

Figure 3

Determination of thermodynamic parameters. (A) The thermodynamic parameters describing the dissociation of probe-target duplexes were determined after measuring the T_m of duplexes that form at variable target concentrations. The determination was performed for triple-stem probe (T) with 17-base PM and 1MM targets. The slope of each fitted line is equal to the negative value of the enthalpy (-ΔH_1→2⁰) and the intercept is equal to the entropy (ΔS_1→2⁰). (B) Free energy change for the three phases of a solution of the triple-stem probe in equilibrium with 17-base PM and 1MM targets. The difference (Φ) between the T_m of PM duplexes (1_PM) and 1MM duplexes (1_1MM) in the triple-stem systems is 43.6 °C.