Investigation Of Newly Identified G-quadruplexes And Its Application To DNA Detection

The G-quadruplexes are guanine-rich nucleic acid sequences that can fold into four-stranded structures. Various small molecules have shown strong affinity to G-quadruplex structures, and the G-quadruplex can generate colorimetric, fluorescence, chemiluminescence signal when complexing with different ligands. Therefore, G-quadruplex structures have attracted intense attention as signal transducers in bioanalytical application. The most commonly used G-quadruplexes are PS2.M, PS5.M and PW17. Nevertheless, it was of great importance to explore new G-quadruplexes to improve the analytical performance of G-quadruplex-based bioassays. In our previous work, two G-rich aptamers,9th-3-35 and 10th-2-40, were obtained. In order to explore their further application, here we investigated the properties of the two sequences. In addition, we proposed a DNA detection platform based on properties of the newly identified G-quadruplex and the enzyme-assisted amplification. What is more, we propsed a DNA detection platform based on split G-quadruplex. The details are summarized as follows:1. The peroxidase-like activity and enhanced ThT fluorescence response of two newly identified G-quadruplexes were investigated. The G-quadruplexes can interact with hemin and the formed G-quadruplex/hemin complexes show peroxidase-like activity that can catalyse the oxidation of 2,2'-azinobis (3-ethylbenzothiozoline-6-sulfonic acid) (ABTS) by H2O2 into coloured products. The interactions between G-quadruplexes and hemin were characterized with UV-vis spectra and circular dichroism (CD) spectra. The peroxidaselike activity of G-quadruplex in ABTS-H2O2 system was studied. The results indicated that 9th-3-35 and 10th-2-40 showed excellent peroxidase-like activity that were both comparable to PS5.M while better than PS2.M and PW17. In addition, fluorescence of ThT could be dramatically enhanced upon binding with the two G-quadruplexes, and the interactions of the G-quadruplexes with ThT were characterized with UV-vis spectra, fluorescence spectra and CD spectra. The results indicated that compared with PS2.M, PS5.M and PW17,9th-3-35 and 10th-2-40 exhibited a more dramatic fluorescence enhancement. This studies indicated that the newly identified G-quadruplexes are competitive candidates that are promising to be utilized as signal transducer to develop G-quadruplex-based bioassays.2. A DNA detection platform was developed based on G-quadruplex enhanced fluorescence of ThT and strand displacement amplification (SDA). Upon hybridization with target DNA, the template is extended and cleaved repeatedly in the presence of polymerase and nicking enzyme, yielding numerous G-quadruplexes that can enhance the fluorescence of ThT. Whereas, SDA reaction could not be triggered in the absence of target DNA, leading to low fluorescence background. We investigated the application of two newly identified G-quadruplexes to DNA detection. The results indicated that the G-quadruplex 9th-3-35 could be applied to sensitive DNA detection with a detection limit of 8 pM, and the designed DNA detection platform could detect target DNA with good selectivity. The results implied that 9th-3-35 was competitive candidate to be used in the design of bioassays.3. The fluorescence enhancement of split G-quadruplexes to ThT was investigated by splitting the G-quadruplex 9th-3-35 into different split modes (mode A-G), and the application of split G-quadruplexes as signal transducers for DNA detection was performed. The G-quadruplex 9th-3-35 was split into two G-rich segments, and the G-rich segments could not form G-quadruplex structure and could not enhance the fluorescence of ThT. Whereas in the presence of target DNA, two G-rich segments were driven together by hybridization to enhance the fluorescence of ThT, so detection of target DNA could be realised. We could choose different split modes based on different analytical purposes:mode G for high sensitvie DNA detection and mode B for single nucleotide polymorphism detection.The use of split G-quadruplexes makes the design of bioassays more flexible and the operation easier. To extend the application of split G-quadruplex, further investigation was commented to be carried out by optimizing the design of probes.