Institut für Chemie der Humboldt-Universität zu Berlin, Brook-Taylor Strasse 2, 12489 Berlin, Germany.
Chembiochem. 2012 Sep 24;13(14):2072-81. doi: 10.1002/cbic.201200397. Epub 2012 Aug 31.
Oligonucleotide hybridization probes that fluoresce upon binding to complementary nucleic acid targets allow the real-time detection of DNA or RNA in homogeneous solution. The most commonly used probes rely on the distance-dependent interaction between a fluorophore and another label. Such dual-labeled oligonucleotides signal the change of the global conformation that accompanies duplex formation. However, undesired nonspecific binding events and/or probe degradation also lead to changes in the label-label distance and, thus, to ambiguities in fluorescence signaling. Herein, we introduce singly labeled DNA probes, "DNA FIT probes", that are designed to avoid false-positive signals. A thiazole orange (TO) intercalator dye serves as an artificial base in the DNA probe. The probes show little background because the attachment mode hinders 1) interactions of the "TO base" in cis with the disordered nucleobases of the single strand, and 2) intercalation of the "TO nucleotide" with double strands in trans. However, formation of the probe-target duplex enforces stacking and increases the fluorescence of the TO base. We explored open-chain and carbocyclic nucleotides. We show that the incorporation of the TO nucleotides has no effect on the thermal stability of the probe-target complexes. DNA and RNA targets provided up to 12-fold enhancements of the TO emission upon hybridization of DNA FIT probes. Experiments in cell media demonstrated that false-positive signaling was prevented when DNA FIT probes were used. Of note, DNA FIT probes tolerate a wide range of hybridization temperature; this enabled their application in quantitative polymerase chain reactions.
寡核苷酸杂交探针在与互补核酸靶标结合时会发出荧光，从而允许在均相溶液中实时检测 DNA 或 RNA。最常用的探针依赖于荧光团和另一个标记之间的距离依赖性相互作用。这种双标记的寡核苷酸信号表明伴随双链体形成的全局构象变化。然而，不希望的非特异性结合事件和/或探针降解也会导致标记-标记距离的变化，从而导致荧光信号的模糊。在此，我们引入了单标记 DNA 探针，“DNA FIT 探针”，旨在避免假阳性信号。噻唑橙（TO）嵌入染料在 DNA 探针中用作人工碱基。探针显示出很少的背景，因为附着模式阻碍了 1）“TO 碱基”在顺式与单链上无规核碱基的相互作用，和 2）“TO 核苷酸”在反式与双链的嵌入。然而，探针-靶标双链体的形成强制进行堆积并增加 TO 碱基的荧光。我们探索了开链和碳环核苷酸。我们表明，TO 核苷酸的掺入对探针-靶标复合物的热稳定性没有影响。当杂交 DNA FIT 探针时，DNA 和 RNA 靶标提供了高达 12 倍的 TO 发射增强。在细胞培养基中的实验表明，当使用 DNA FIT 探针时，防止了假阳性信号。值得注意的是，DNA FIT 探针可以容忍广泛的杂交温度；这使其能够在定量聚合酶链反应中应用。
