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.

Impedance-Based DNA Biosensor Employing Molecular Beacon DNA as Probe and Thionine as Charge Neutralizer

“…The target sequences were all 1.0 Â 10 À6 mol L [16,19], the data were 55.6% and 51%. The enhanced ability of this biosensor to distinguish the target DNA sequences was attributed to the hairpin structure in the beacon DNA probe [27].…”

“…The ssDNA probe used in the experiments belongs to molecular beacon, a class of single-stranded oligonucleotides possessing a stem-and-loop structure and having high base pair mismatch identification capability [27]. Traditionally, molecular beacon-based DNA biosensors employ fluorescence detection techniques by linking a fluorophore and a quencher molecule onto the each end of the stem [28].…”

“…Because the density of unstructured probe on electrode surface was nearly twice of that of hairpin one [27], in this work the surface coverage of beacon ssDNA probe on Au electrode was estimated from that of unstructured one by using an electrochemical measurement [42]. Briefly, methylene blue (MB) reacts with guanine bases of ssDNA specially [42].…”

Optimization Strategies for DNA Microarray-Based Detection of Bacteria with 16S rRNA-Targeting Oligonucleotide Probes

“…In recent years, several systematic studies were conducted on different aspects, such as the secondary structures of target molecules and steric hindrance mediated by the solid support (8,22,33,40,42,49), but the complex interaction of all these mechanisms and their influence on hybridization specificity are still mostly unknown. In this study, we systematically evaluated the suitability of the microarray format for 16S rRNA-based detection of bacteria by analyzing a model system including single-mismatch controls.…”

LidNA, a novel miRNA inhibitor constructed with unmodified DNA

“…Variations 15 and 16 were constructed with the miRNA binding site and the doublestranded region at 5 0 terminal and they were less effective than variations 12, 13, and 14 with miRNA binding sites and doublestranded regions at the 3 0 terminal. A previous report shows that hairpin-structured DNA probes bound anti-sense strands with higher affinity than liner singlestranded DNA probes [20]. In this study, we designed an experiment to measure the affinity of LidNA-type probe for miRNA using Biacore 3000 (GE Healthcare).…”