Riccelli P V, Merante F, Leung K T, Bortolin S, Zastawny R L, Janeczko R, Benight A S
Department of Chemistry, University of Illinois at Chicago, IL 60607, USA.
Nucleic Acids Res. 2001 Feb 15;29(4):996-1004. doi: 10.1093/nar/29.4.996.
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.
本文描述了一种基于微量滴定板的检测系统，其中固定了带有悬垂末端的DNA发夹探针和单链线性DNA探针，并根据它们捕获单链靶DNA的能力进行比较。发夹探针由一个16bp的双链茎组成，通过T(2)-生物素.dT-T(2)环连接。第三个碱基是与抗生物素蛋白包被的微量滴定孔偶联所必需的生物素化尿嘧啶（U(B)）。发夹的捕获区域是一个由16或32个碱基组成的3'悬垂末端。对该系统的基本参数，如探针密度和平板的抗生物素蛋白吸附能力进行了表征。靶DNA由65个碱基组成，其3'末端与发夹的悬垂末端或线性探针序列互补。该检测系统用于测量靶标与发夹探针和线性探针杂交的时间依赖性和热力学稳定性。靶分子用5'-FITC标记，或用[γ-(33)P]ATP进行放射性标记，并被固定在固体支持物上的线性或发夹探针捕获。在10至640pmol的靶标浓度范围内，杂交率随靶标浓度的增加而增加，但不同检测探针的杂交率有所不同。与线性探针相比，发夹探针显示出更高的杂交率和更大的捕获靶标平衡量。在25和45℃下，发夹探针的杂交率比线性捕获探针高出两倍以上。发夹-靶标复合物在热力学上也更稳定。根据观察到的确形成复合物的平衡常数评估结合自由能。结果表明探针的稳定性顺序为：具有32个碱基悬垂末端的发夹>具有16个碱基悬垂末端的发夹探针>16个碱基的线性探针>32个碱基的线性探针。发夹的物理特性作为基于固体支持物的杂交系统中的核酸捕获部分可能具有显著优势。
