Svanvik N, Westman G, Wang D, Kubista M
Department of Molecular Biotechnology, Lundberg Institute, Göteborg, Sweden.
Anal Biochem. 2000 May 15;281(1):26-35. doi: 10.1006/abio.2000.4534.
We have constructed light-up probes for nucleic acid detection. The light-up probe is a peptide nucleic acid (PNA) oligonucleotide to which the asymmetric cyanine dye thiazole orange (TO) is tethered. It combines the excellent hybridization properties of PNA and the large fluorescence enhancement of TO upon binding to DNA. When the PNA hybridizes to target DNA, the dye binds and becomes fluorescent. Free probes have low fluorescence, which may increase almost 50-fold upon hybridization to complementary nucleic acid. This makes the light-up probes particularly suitable for homogeneous hybridization assays, where separation of the bound and free probe is not necessary. We find that the fluorescence enhancement upon hybridization varies among different probes, which is mainly due to variations in free probe fluorescence. For eight probes studied the fluorescence quantum yield at 25 degrees C in the unbound state ranged from 0.0015 to 0.08 and seemed to depend mainly on the PNA sequence. The binding of the light-up probes to target DNA is highly sequence specific and a single mismatch in a 10-mer target sequence was readily identified.
我们构建了用于核酸检测的发光探针。该发光探针是一种肽核酸（PNA）寡核苷酸，不对称花青染料噻唑橙（TO）连接在其上。它结合了PNA优异的杂交特性以及TO与DNA结合后荧光大幅增强的特性。当PNA与靶DNA杂交时，染料结合并发出荧光。游离探针荧光较低，与互补核酸杂交后荧光可能增加近50倍。这使得发光探针特别适用于均相杂交分析，无需分离结合态和游离态探针。我们发现不同探针杂交后的荧光增强有所不同，这主要是由于游离探针荧光的差异。对于所研究的8种探针，25℃下未结合状态的荧光量子产率在0.0015至0.08之间，似乎主要取决于PNA序列。发光探针与靶DNA的结合具有高度序列特异性，很容易识别10聚体靶序列中的单个错配。
