Institute of Physical Chemistry, Zhejiang Normal University, Jinhua 321004, Zhejiang, People's Republic of China.
Analyst. 2011 Nov 7;136(21):4480-5. doi: 10.1039/c1an15652g. Epub 2011 Sep 22.
DNA single-nucleotide polymorphism (SNP) detection has attracted much attention due to mutation-related diseases. Various fluorescence methods for SNP detection have been proposed and many are already in use. However, fluorescence enhancement for signal-on SNP identification without label modification still remains a challenge. Here, we find that the abasic site (AP site) in a DNA duplex can be developed as a binding pocket favorable for the occurrence of the excited-state intramolecular proton transfer (ESIPT) of a 3-hydroxyflavone, fisetin, which is used as a proof of concept for effective SNP identification. Fisetin binding at the AP site is highly selective for target thymine or cytosine facing the AP site by observation of a drastic increase in the ESIPT emission band. In addition, the target recognition selectivity based on this ESIPT process is not affected by flanking bases of the AP site. The binding selectivity of fisetin at the AP site is also confirmed by measurements of fluorescence resonance energy transfer, emission lifetime and DNA melting. The fluorescent signal-on sensing for SNP based on this fluorophore is substantially advantageous over the previously used fluorophores such as the AP site-specific signal-off organic ligands with a similar fluorescing mechanism before and after binding to DNA with hydrogen bonding interaction. We expect that this approach will be employed to develop a practical SNP detection method by locating an AP site toward a target and employing an ESIPT probe as readout.
由于与突变相关的疾病，DNA 单核苷酸多态性 (SNP) 的检测引起了广泛关注。已经提出了各种用于 SNP 检测的荧光方法，并且其中许多已经在使用。然而，无需标记修饰即可进行用于信号开启 SNP 识别的荧光增强仍然是一个挑战。在这里，我们发现 DNA 双链体中的无碱基位点 (AP 位点) 可以被开发为一个结合口袋，有利于 3-羟基黄酮，非瑟酮的激发态分子内质子转移 (ESIPT) 的发生，这被用作有效 SNP 识别的概念验证。通过观察到 AP 位点对面的靶胸腺嘧啶或胞嘧啶的 ESIPT 发射带急剧增加，非瑟酮在 AP 位点的结合具有高度的选择性。此外，基于该 ESIPT 过程的靶识别选择性不受 AP 位点侧翼碱基的影响。通过荧光共振能量转移、发射寿命和 DNA 熔解的测量，还证实了非瑟酮在 AP 位点的结合选择性。与以前使用的与氢键相互作用与 DNA 结合前后具有相似荧光机制的 AP 位点特异性信号关闭有机配体等荧光团相比，基于这种荧光团的 SNP 的荧光信号开启感应具有显著优势。我们期望通过将 AP 位点定位到靶标并将 ESIPT 探针用作读出，采用这种方法来开发实用的 SNP 检测方法。
