Screening Of G-quadruplex Ligand Of The Fluorescence Anisotropy Method

The single-stranded DNA at 3'end of telomere is a guanine-rich sequence, which can form G-quadruplexes under certain conditions by Hoogsteen hydrogen bonding. G-quadruplexes are DNA secondary structure, which can effectively inhibit the activity of telomerase. Therefore, compounds that are capable of inducing and stabilizing the G-quadruplex structure has potential anti-tumor effect. Selection of G-quadruplex-binding ligands is of great theoretical and practical importance for the research and design of anti-tumor drugs. In this thesis, fluorescence anisotropy methods have been developed to simply and rapidly select quadruplex-binding ligands from natural anticancer drugs based on anisotropy change of F-GDNA induced by structure-switching of fluorescent dye-labeled human telomere. The contents and results of research in this thesis contain the following three parts:1. Selecting G-quadruplex-binding ligands using noncompetitive fluorescence anisotropyA fluorescence anisotropy method has been proposed to identify natural drugs that stabilize G-quadruplexes in a homogeneous medium using fluorescein-tagged human telomeric DNA sequence (F-GDNA) as nucleic acid probe. The fluorescence anisotropy of single-stranded F-GDNA is low. However, the fluorescence anisotropy of F-GDNA increased in the presence of ligands which can induce the F-GDNA to form G-quadruplexes because of limited moving of fluorescent dye. Seven kinds of natural anticancer drugs were studied by the proposed method. The combined data from fluorescence anisotropy and CD measurements indicated that it is a simple, fast and effective approach to identify potential G-quadruplex ligands.2. Selecting G-quadruplex-binding ligands using competitive fluorescence anisotropyA competitive fluorescence anisotropy method has been proposed to identify natural drugs that stabilize G-quadruplexes in a homogeneous medium using fluorescein-tagged human telomeric DNA (F-GDNA) as nucleic acid probe. In the absence of GDNA, F-GDNA can be induced into G-quadruplex by aloe-emodin. The local motional freedom of the FAM is restricted and the fluorescence anisotropy signal of F-GDNA/aloe-emodin is larger. In the presence of GDNA, the competition interaction of F-GDNA and GDNA replaced part of F-GDNA by GDNA, resulting in the decrease of the fluorescence anisotropy of the sensing system. Twelve anticancer drugs were investigated as potential ligands using proposed method. The formation of G-quadruplex has been verfied by CD measurements. The combined data indicated that it is a simple and effective approach to identify potential G-quadruplex ligands.3. Selecting G-quadruplex-binding ligands based on the competition of ligands and complementary strands of telomere DNAA competitive fluorescence anisotropy method has been estabilished for fast identifying G-quadruplex ligands based on the competition of complementary strands of telomere DNA and G-quadruplex ligands. The fluorescence anisotropy of F-GDNA/cDNA hybrid is larger because the local motional freedom of the FAM is restricted. In the presence of G-quadruplex-binding ligands, the competition interaction between G-quadruplex-binding ligands and complementary strands (cDNAs) of telomere DNA released part of F-GDNA from F-GDNA/cDNA hybrid, which reduced the fluorescence anisotropy of F-GDNA/cDNA hybid. The competition interactions between berberine and complementary strands of telomere DNA with distinct sequence length have been studied. The combined data from fluorescence anisotropy and CD measurements indicated that ligands selected by this fluorescence anisotropy could induce F-GDNA to fold into G-quadruplex and destabilize the Watson-Crick duplexes of F-GDNA/cDNA. This fluorescence anisotropy method offers a simple and effective approach to identify ligands with potential anticancer activity.