Tsourkas Andrew, Behlke Mark A, Rose Scott D, Bao Gang
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Nucleic Acids Res. 2003 Feb 15;31(4):1319-30. doi: 10.1093/nar/gkg212.
Molecular beacons are increasingly being used in many applications involving nucleic acid detection and quantification. The stem-loop structure of molecular beacons provides a competing reaction for probe-target hybridization that serves to increase probe specificity, which is particularly useful when single-base discrimination is desired. To fully realize the potential of molecular beacons, it is necessary to optimize their structure. Here we report a systematic study of the thermodynamic and kinetic parameters that describe the molecular beacon structure-function relationship. Both probe and stem lengths are shown to have a significant impact on the binding specificity and hybridization kinetic rates of molecular beacons. Specifically, molecular beacons with longer stem lengths have an improved ability to discriminate between targets over a broader range of temperatures. However, this is accompanied by a decrease in the rate of molecular beacon-target hybridization. Molecular beacons with longer probe lengths tend to have lower dissociation constants, increased kinetic rate constants, and decreased specificity. Molecular beacons with very short stems have a lower signal-to-background ratio than molecular beacons with longer stems. These features have significant implications for the design of molecular beacons for various applications.
分子信标越来越多地用于许多涉及核酸检测和定量的应用中。分子信标的茎环结构为探针-靶标杂交提供了一种竞争反应，有助于提高探针特异性，这在需要单碱基区分时特别有用。为了充分发挥分子信标的潜力，有必要优化其结构。在此，我们报告了一项关于描述分子信标结构-功能关系的热力学和动力学参数的系统研究。结果表明，探针和茎的长度对分子信标的结合特异性和杂交动力学速率都有显著影响。具体而言，茎长度较长的分子信标在更宽的温度范围内具有更强的区分靶标的能力。然而，这伴随着分子信标-靶标杂交速率的降低。探针长度较长的分子信标往往具有较低的解离常数、增加的动力学速率常数和降低的特异性。茎非常短的分子信标比茎较长的分子信标具有更低的信噪比。这些特性对设计用于各种应用的分子信标具有重要意义。
