Studies On Single Nucleotide Polymorphism Biosensing Based On Excited-state Intramolecular Proton Transfer Probe

Single nucleotide polymorphism (SNP) mainly refers to the level of the genome sequence polymorphism caused by a single nucleotide variations. SNP is one of the types of mutations that cause common diseases and becomes the most common type of human genetic variation.Therefore, the research of SNP has become a hot topic in the era of genome. In this thesis, the detection method of SNP based on DNA abasic site (AP site) has many advantages, such as rapidity, sensitivity and low background. On this basis, we have successfully developed the detection method of flavonoids that using the AP-DNA as aptamer. The main contents are as follows:1. Study on fluorescence light-up recognition of single-nucleotide polymorphism based on selective of abasic site binding of Fisetin probeWe 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 a3-hydroxyflavone, fisetin, which is used as a proof of concept for fluorescence light-up identification of SNP. The AP site was deliberately introduced in a probe strand that opposite the target base favorable for the occurrence of the excited-state intramolecular proton transfer (ESIPT) of the fisetin. However, the essiciency of ESIPT depends on the target base just opposite the AP site. Selective binding of Fisetin at AP site was found by recognition of SNP. The results show that:when the target base opposite the AP site is cytosine or thymine, the maximum enhancement of Fisetin fluorescence emission can be obtained. However, the target base is guanine or adenine, minor fluorescence emission changes are observed. This method benefits from signal-on SNP detection with high selectivity by differentiation of pyrimidine from purine. In particular, this differentiation of target base is not influenced by the flanking bases of the AP site. Innovation of this approach is developed to a un-labled fluorescence light-up recognition of SNP.2. Study on DNA abasic site-based aptamer for detection of flavonoidsThe abasic site-containing DNAs (AP-DNAs) were employed as aptamer, the aptamer exhibits high selectivity and sensitivity for binding of fisetin over the other flavonoids such as morin, rutin, apigenin, kaempferol, myricetin, quercetin, fisetin, luteolin, baicalin, naringenin, genistein, chrysin, and galangin. This selective binding will be employed to develop a rapid and practical detection of Fisetin. The results show that:The detection limit is about50nM at a signal-to-noise ratio of3. Aptamer designed in this method without any fluorophore modification. Thus, the almost negligible fluorescence background from the aptamer is achieved.3. Study on the recognition of AP sites based on targeting DNA abasic site by myricetinIn this study, one of natural3-hydroxyflavonols, myricetin (Myr), was employed as probe, The binding specificity were confirmed by the methods of steady-state and transient-state fluorescence, FRET, and DNA melting experiments. The results show that: Myr alone in aqueous solution is non-fluorescent, However, the AP site binding of Myr favors an emission from its tautomer that is derived from excited-state intramolecular proton transfer (ESIPT) reaction between the3-OH and4-carbonyl moieties. More importantly, the selective recognition of Myr is less dependent on the flanking bases of the AP site. Thus, this selective lighting-up emission is advantageous for developing a practical sensor to target the DNA AP site with a weak fluorescence background. This selective recognition of the AP site by the lighting-up fluorescence response would find wide applications including efficiently evaluating DNA damage/repair and screening antitumor/antioxidation drugs.