A New Method For Studying Telomerase Activity And Inhibition Of Fluorescence

Telomerase activity is overexpressed in more than 85% of tumor cells but not in normal somatic cells. Thus, as a kind of universal tumor marker, the research on the telomerase activity and inhibition is particularly important in the telomerase-targeted diagnosis and treatment of tumors. G-quadruplex formation at the end of telomere could effectively inhibit the telomerase activity and curb the proliferation of tumor cells. Therefore, it is important to screen and discover G-quadruplex binding ligands which could induce human telomere GDNA to form G-quadruplex for designing telomerase-targeted anti-tumor drugs. In this thesis, new fluorescence methods have been developed for studying telomerase activity and screening G-quadruplex binding ligands based on the conformation transfer induced by molecular recognition.The main content is summarized as the following three parts.1. PCR-free and sensitive detection of telomerase activity based on photo-induced electron transferA PCR-free fluorescent method was developed for ultra-sensitively monitoring activity and inhibition of human telomerase based on photo-induced electron transfer (PIET).A FAM-modified oligonucleotide probe (FDNA) served as the fluorescent signal probe. In the presence of telomerase, the 3’end of telomerase substrate (TS) primer was elongated to form a long single-stranded DNA containing the human telomere repeat units (TTAGGG). This long DNA could hybridize with numerous of FDNA, which led the fluorophore labeled on FDNA just to be close to GGG bases of human telomere repeat sequence accompanying with the fluorescence quenching of FDNA due to PEET between deoxyguanosine and fluorophore. Because the long elongation product can hybridize with multiple FDNA for amplifying the fluorescent quenching efficiency, telomerase activity was sensitively detected without the polymerase chain reaction (PCR) amplification and enzyme-aided signal amplification. The telomerase activity equivalent to 1 HeLa cell could be detected by this method.2. Identifying G-Quadruplex-Binding Ligands via Conformational Changes-Induced Fluorescence Quenching of DNA/Silver Nanocluster Herein, a label-free and luminescent switch-off strategy was designed for identifying G-quadruplex-binding ligands using DNA/silver nanocluster (DNA-AgNC) as signal probe. It is based on the fact that guanines (G) and G-quadruplex show different influence on the fluorescence intensity of DNA-AgNC. The designed probe integrates G-rich human telomeric DNA (GDNA, for recognizing ligand), template sequence (for in-situ synthesis of AgNC) and thymine-rich linker. The fluorescence of in-situ synthesized DNA-AgNC was strengthened by GDNA. Whereas, an obvious fluorescence quenching was observed as a result of G-quadruplex information induced by ligand. Thus, the G-quadruplex-binding ligands can be specifically screened by monitoring DNA-AgNC fluorescence changes. The fluorescence, UV-Vis absorption spectrum and transmission electron microscopy demonstrate that the synthesized AgNC has well fluorescence properties and uniform size. Circular dichroism and binding constants measurement verified that the selected ligand can indeed induce the formation of G-quadruplex and bind the telomere GDNA in a relative stable manner, respectively. Fluorescence imaging and MTT assay demonstrated that ligands can inhibit cell proliferation and induce a massive apoptosis. Therefore, it holds considerable potential in identifying G-quadruplex-binding ligands and discovering telomerase-targeted anticancer drugs.3. Label-free detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplificationA label-free method is developed for detection of T4 polynucleotide kinase (T4 PNK) activity by coupling DNA strand displacement reaction with enzyme-aided amplification. The stem of hairpin oligonucleotide (hpDNA) was phosphorylated by T4 PNK and further degraded by lambda exonuclease (λ exo) from 5’to 3’direction to release a single-stranded DNA to trigger strand displacement reaction(SDR). P2 displaced by the trigger from P1/P2 hybrid can be induced by potassium ions to form G-quadruplex and NMM specifically binds to G-quadruplex resulting in a strong fluorescence. Upon the cleavage of nicking endonuclease (Nt.BbvCI), the trigger can be circularly liberated to displace P2 yielding fluorescence signal amplification for determining T4 PNK activity with a detection limit of 6.6×10-4 U/mL. T4 PNK activity can be studied in both pure and complex homogeneous solution. Moreover, it also can be employed to screen T4 PNK inhibitors. Therefore, this developed strategy provides a new fluorescence method for simple, sensitive and cost-effective analysis of T4 PNK activity and inhibition.