Design And Application Of Fluorescent Aptamer Probes For Angiogenin Analysis

Aptamers are a novel class of synthetic DNA/RNA oligocleotides generated from in vitro selection to bind with proteins and various targets. Due to their high affinity and specificity, easy synthesis, conveniently functional modification and good stability, aptamers have shown great prospect for basic biomedical research and clinical diagnosis and therapy. In this dissertation, angiogenin, which is closely correlated with the occurrence and development of cancers, was chosen as the target protein. Aiming at the design of fluorescent aptamer probes and their applications to angiogenin analysis, a series of angiogenin assays based on aptamers and fluorescent analyses have been developed. The research work of this dissertation is summarized as follows:(1) Aptamer/nucleic acid"molecular light switch"complex probe was designed and synthesized, and a method for angiogenin detection based on aptamer and nucleic acid"molecular light switch"[Ru(bipy)2(dppz)]2+ was developed. This method took advantage of the obvious fluorescent signal decrease of aptamer/nucleic acid"molecular light switch"complex probe upon specific angiogenin/aptamer binding. By monitoring the change of fluorescent intensity, angiogenin detection could be achieved quickly. This assay could determine angiogenin in the range of 2.5~20 nmol/L with a detection limit of 1.5 nmol/L. In this assay, the design of probe is convenient since neither the aptamers nor target proteins should be labeled directly.(2) A singly fluorophore-labeled aptamer probe was designed and synthesized, and a method for angiogenin detection based on fluorescence anisotropy was developed. This method took advantage of the obvious fluorescence anisotropy signal increase of fluorophore-labelled aptamer probe upon specific angiogenin/aptamer binding. By monitoring the anisotropy change, angiogenin detection could be achieved specifically. This assay could determine angiogenin in the range of 2~55 nmol/L with a detection limit of 1 nmol/L. Angiogenin in serum samples from lung cancer patients have also been detected by using this method.(3) A dually fluorophore-labeled aptamer probe was designed and synthesized and a method for angiogenin detection based on fluorescence resonance energy transfer (FRET) was developed. This method took advantage of the FRET efficiency increase caused by configuration shift upon specific protein/aptamer binding. By monitoring the fluorescence intensity of donor and acceptor fluorophores and calculating the FRET efficiency, angiogenin detection could be achieved with high sensitivity. This assay could determine angiogenin in the range of 0.5~40 nmol/L with a detection limit of 0.2 nmol/L. Angiogenin in serum samples from health people and lung cancer patients have also been detected by using this method.(4) A dually pyrene-labeled aptamer probe was designed and synthesized and a method for angiogenin detection based on pyrene excimer was developed. The method took advantage of the obvious fluorescence signal change of excimer caused by configuration shift and encounter of the pyrene molecules at the both termini upon specific protein/aptamer binding. By monitoring the fluorescence intensity of aptamer probe, quantitative angiogenin detection could be achieved with high specificity and sensitivity. This assay could determine angiogenin in the range of 0.2~30 nmol/L with a detection limit of 0.1 nmol/L.(5) The applications of aptamer probes were extended from convenient, quick, highly sensitive and specific protein detection to real-time protein assay in live cells. A novel simple and quick method for visualizing the real-time cellular internalization process of angiogenin was developed by using singly fluorophore-labeled aptamer probe and confocal laser scanning microscopy. Specifically, when aptamer/angiogenin conjugates were added into cell cultures, conjugates could be selectively bound to human umbilical vein endothelial cells and human breast cancer MCF-7 cells. Nuclear staining and Z-axis scanning studies demonstrated that the aptamer/angiogenin conjugates were internalized to intracellular organelles of target cells, and dynamic confocal imaging studies indicated that the conjugates were quickly bound to target cells and internalized. Different temperature culture studies validated that the internalization of aptamer/angiogenin conjugates were dependent on culture temperature and implied an endocytosis process. This method provides the first evidence that a fluorophore-labeled aptamer could be used not only as a protein labeling agent but also as a protein recognition agent to report the actual interaction of proteins with cancer cells in real time without target protein prelabelling, target gene cloning and target cell fixing. This method also provides the promising potential of fluorophore-labeled aptamers used as recognition and labeling probes for tracking and localizing of some important biomolecules such as cell factors, growth factors, polypeptides and enzymes secreted by metabolic processes.The above fluorescent aptamer probe-based analytical methods are convenient and low-cost without using antibody. Angiogenin in serum and living cells could be analysed quickly, sensitively and selectively. Therefore, these aptamer-based methods have the potential to be used in basic research and clinical diagnosis concerning angiogenin.