Hybridization of single-stranded DNA targets to immobilized complementary DNA probes: comparison of hairpin versus linear capture probes

Abstract

A microtiter-based assay system is described in which DNA hairpin probes with dangling ends and single-stranded, linear DNA probes were immobilized and compared based on their ability to capture single-strand target DNA. Hairpin probes consisted of a 16 bp duplex stem, linked by a T(2)-biotin.dT-T(2) loop. The third base was a biotinylated uracil (U(B)) necessary for coupling to avidin coated microtiter wells. The capture region of the hairpin was a 3' dangling end composed of either 16 or 32 bases. Fundamental parameters of the system, such as probe density and avidin adsorption capacity of the plates were characterized. The target DNA consisted of 65 bases whose 3' end was complementary to the dangling end of the hairpin or to the linear probe sequence. The assay system was employed to measure the time dependence and thermodynamic stability of target hybridization with hairpin and linear probes. Target molecules were labeled with either a 5'-FITC, or radiolabeled with [gamma-(33)P]ATP and captured by either linear or hairpin probes affixed to the solid support. Over the range of target concentrations from 10 to 640 pmol hybridization rates increased with increasing target concentration, but varied for the different probes examined. Hairpin probes displayed higher rates of hybridization and larger equilibrium amounts of captured targets than linear probes. At 25 and 45 degrees C, rates of hybridization were better than twice as great for the hairpin compared with the linear capture probes. Hairpin-target complexes were also more thermodynamically stable. Binding free energies were evaluated from the observed equilibrium constants for complex formation. Results showed the order of stability of the probes to be: hairpins with 32 base dangling ends > hairpin probes with l6 base dangling ends > 16 base linear probes > 32 base linear probes. The physical characteristics of hairpins could offer substantial advantages as nucleic acid capture moieties in solid support based hybridization systems.

Figure 1

DNA sequences and schematics of the probe DNA molecules. Hairpin sequences (a) and (b) were designed to form intramolecular stem–loop structures with 3′ dangling ends with sequences complementary to the 3′ end of the target strand. Hairpins also contained a biotinylated uracil at the third base position in the loop for coupling to an avidin-coated microtiter plate. Hairpins have the same 16 bp duplex stems and either 32 (a) or 16 (b) base dangling ends. Linear probes were 32 (c) and 16 (d) bases in length. Biotin derived bases contained a 12-atom spacer at the 5′ end for coupling to plates. Target strand (e) corresponded to a 65 base region of the porcine malignant hyperthermia gene from positions 958 to 1022.

Figure 2

Schematic of the ERV. Dimensions of the ERV are shown. The upper limit of the ERV is defined by the reactive surface area of the plate well covered by 100 µl and estimated height (h) of the 32 base hairpin capture probe coupled to the surface. For our system the surface area of the plate well covered by 100 µl was estimated to be 0.9482 cm², and the height of the biotinylated 32 base hairpin probe was estimated to be 250 Å. These values result in an estimated ERV of 2.371 × 10^–3 µl.

Figure 3

Coating of microtiter plates with avidin. Results of avidin-coating experiments are shown for two different detection methods. The amount of avidin required to saturate a microwell is plotted versus the amount of FITC–avidin added to the wells with increasing concentrations of FITC–avidin until saturation as shown. (a) Relative fluorescence units (RFU) determined by fluorometry of bound avidin. Error bars denote the SD of at least four independent coating experiments. (b) FITC–avidin binding curves determined indirectly by an anti-FITC alkaline phosphatase antibody and chemiluminescence detection.

Figure 4

Coupling of probes to the plate surface. Standard curves derived for each type of DNA probe where the amounts of biotinylated linear and hairpin probes bound to the surface are plotted against the amounts of probe added in 100 µl. Error bars are SDs of at least four independent experiments. Clearly, for the different types of probes the amounts coupled to the avidin wells increase with increasing amounts of added probe, but the amount of bound probe is not the same for all probes.

Figure 5

Relative binding as a function of input target concentration. The summed RLU (net RLU), corresponding to captured FITC-labeled target strand, is plotted against the amount of target strand added for each type of probe. The 32 base hairpin (triangles) captured the highest quantity of FITC-target strand. At somewhat lesser amounts are the 16mer hairpins (circles), 32 base linear (diamonds) and 16 base linear (squares) probes. The avidin-coated wells were coated with 5 × 10^–8 M probe strands.

Figure 6

Probe–target complex formation as a function of time. Plots of the concentration of target–probe complexes, [P*T], versus time for four target concentrations at 45°C are shown for each probe. (a) 32mer hairpin, (b) 16mer hairpin, (c) 32mer linear, (d) 16mer linear. Solid lines drawn through the data are best fits obtained from non-linear least square regression fits of the data with equation 6.