Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resources Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China.
Chemistry. 2011 Feb 1;17(5):1635-41. doi: 10.1002/chem.201001331. Epub 2011 Jan 5.
We demonstrate a novel concept for the construction of a label-free, quadruplex-based functional molecular beacon (LFG4-MB) by using G-quadruplex motif as a substitute for Watson-Crick base pairing in the MB stem and a specific G-quadruplex binder, N-methyl mesoporphyrin IX (NMM) as a reporter. It shows high sensitivity in assays for UDG activity/inhibition and detection of DNA sequence based on the unique fluorescence increase that occurs as a result of the strong interaction between NMM and the folded quadruplex upon removal of uracil by UDG or displacement of block sequence by target DNA. The LFG4-MB is simple in design, fast in operation and could be easily transposed to other biological relevant target analysis by simply changing the recognition portion. The LFG4-MB does not require any chemical modification for DNA, which offers the advantages of simplicity and cost efficiency and obviates the possible interference with the affinity and specificity of the MB as well as the kinetic behavior of the catalysts caused by the bulky fluorescent groups. More importantly, the LFG4-MB offers great extent of freedom to tune the experimental conditions for the general applicability in bioanalysis.
我们展示了一种新的概念，用于构建无标记的四链体基功能分子信标（LFG4-MB），方法是将 G-四链体基序用作 MB 茎中的 Watson-Crick 碱基配对的替代品，并用特定的 G-四链体结合物 N-甲基甲川卟啉 IX（NMM）作为报告分子。它在 UDG 活性/抑制测定和基于序列的 DNA 检测中表现出高灵敏度，这是由于 UDG 去除尿嘧啶或靶 DNA 置换封锁序列后，NMM 与折叠四链体之间发生强相互作用导致的独特荧光增加所致。LFG4-MB 设计简单，操作快速，只需简单改变识别部分即可轻松转换为其他生物相关目标分析。LFG4-MB 不需要对 DNA 进行任何化学修饰，这具有简单和高效成本的优点，并避免了可能由于庞大的荧光基团而对 MB 的亲和力和特异性以及催化剂的动力学行为产生的干扰。更重要的是，LFG4-MB 为在生物分析中的普遍适用性提供了很大的调节实验条件的自由度。
