Tsourkas Andrew, Behlke Mark A, Bao Gang
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 315 Ferst Drive, Suite 2306, Atlanta, GA 30332, USA.
Nucleic Acids Res. 2002 Oct 1;30(19):4208-15. doi: 10.1093/nar/gkf536.
Molecular beacons are oligonucleotide probes capable of forming a stem-loop hairpin structure with a reporter dye at one end and a quencher at the other end. Conventional molecular beacons are designed with a target-binding domain flanked by two complementary short arm sequences that are independent of the target sequence. Here we report the design of shared-stem molecular beacons with one arm participating in both stem formation when the beacon is closed and target hybridization when it is open. We performed a systematic study to compare the behavior of conventional and shared-stem molecular beacons by conducting thermodynamic and kinetic analyses. Shared-stem molecular beacons form more stable duplexes with target molecules than conventional molecular beacons; however, conventional molecular beacons may discriminate between targets with a higher specificity. For both conventional and shared-stem molecular beacons, increasing stem length enhanced the ability to differentiate between wild-type and mutant targets over a wider range of temperatures. Interestingly, probe-target hybridization kinetics were similar for both classes of molecular beacons and were influenced primarily by the length and sequence of the stem. These findings should enable better design of molecular beacons for various applications.
分子信标是一种寡核苷酸探针，能够形成茎环发夹结构，一端带有报告染料，另一端带有淬灭剂。传统的分子信标设计有一个靶标结合结构域，两侧是两个与靶标序列无关的互补短臂序列。在此，我们报告了共享茎分子信标的设计，当信标关闭时，其中一个臂参与茎的形成，当信标打开时，参与靶标杂交。我们进行了一项系统研究，通过热力学和动力学分析来比较传统分子信标和共享茎分子信标的行为。共享茎分子信标与靶标分子形成的双链体比传统分子信标更稳定；然而，传统分子信标可能对靶标的区分具有更高的特异性。对于传统分子信标和共享茎分子信标，增加茎的长度都增强了在更宽温度范围内区分野生型和突变型靶标的能力。有趣的是，两类分子信标的探针 - 靶标杂交动力学相似，并且主要受茎的长度和序列影响。这些发现应该能够为各种应用更好地设计分子信标。
