Sando Shinsuke, Abe Hiroshi, Kool Eric T
Department of Chemistry, Stanford University, Stanford, California 94305-5080, USA.
J Am Chem Soc. 2004 Feb 4;126(4):1081-7. doi: 10.1021/ja038665z.
We describe the synthesis and study of multicolor quenched autoligating (QUAL) probes for identification and discrimination of closely related RNA and DNA sequences in solution and in bacteria. In these probes, a dabsyl quencher doubles as an activator in the oligonucleotide-joining reaction. The oligonucleotides remain dark until they bind at adjacent sites, and "light up" on nucleophilic displacement of the dabsyl probe by the phosphorothioate probe. Four fluorescent dye conjugates were prepared and tested with probes and targets that differ by one nucleotide. Experiments on polymer beads show clear color-based discrimination of DNAs added in solution. Two-color quenched probe pairs were then tested in the discrimination of 16S rRNA sequences in Escherichia coli. Single nucleotide resolution was achieved in the cells with green/red QUAL probes, allowing identification of a one-base sequencing error in the 16S rRNA database. Finally, QUAL probes were successfully applied in live bacterial cells. The method requires only incubation followed by fluorescence imaging, and requires no enzymes, added reagents, cross-linking, fixing, or washes. Because probes must bind side-by-side to generate signal, there is little or no interference from unintended protein binding, which can occur with other probe types. The results suggest that QUAL probes may be of general use in the detection and identification of sequences in solution, on microarrays, and in microorganisms.
我们描述了用于识别和区分溶液及细菌中密切相关的RNA和DNA序列的多色淬灭自连接（QUAL）探针的合成与研究。在这些探针中，一个达布磺酰淬灭剂在寡核苷酸连接反应中兼作激活剂。寡核苷酸在相邻位点结合之前一直保持暗态，而在硫代磷酸酯探针将达布磺酰探针亲核取代时“点亮”。制备了四种荧光染料缀合物，并与相差一个核苷酸的探针和靶标进行了测试。在聚合物珠上进行的实验表明，能够基于颜色清晰地区分溶液中添加的DNA。然后用双色淬灭探针对在大肠杆菌中鉴别16S rRNA序列进行了测试。使用绿色/红色QUAL探针在细胞中实现了单核苷酸分辨率，从而能够识别16S rRNA数据库中的一个单碱基测序错误。最后，QUAL探针成功应用于活细菌细胞。该方法仅需孵育后进行荧光成像，无需酶、添加试剂、交联、固定或洗涤。由于探针必须并排结合才能产生信号，因此几乎不会受到其他类型探针可能出现的意外蛋白质结合的干扰。结果表明QUAL探针可能在溶液、微阵列及微生物中序列的检测和识别方面具有广泛用途。
