Selection And Identification Of Cancer-specific Aptamers And Their Preliminary Application

Aptamers are short single stranded DNA or RNA molecules, which are evolved from an in vitro selection process known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment), a high-flux screening technique that involves the progressive selection of highly specific ligands by repeated rounds of selection and amplification from a large random synthetic nucleic acid library. They have the ability to bind specifically to their targets, ranging from small organic molecules to complex proteins, based on their specific tertiary structures. Due to the properties of precise discrimination, easy synthesis and modification, less immunogenicity and small size, aptames are emerging candidates for medical research and application. The recently developed complex target SELEX even generates aptamers that differentiate target cells from counter-selected partners, without the prior knowledge of the molecular differences. It provides a new approach for molecular probes development and biomarker verification by the obtained specific aptamers. In this study, we improved the complex target selection method and widen its use in tumor diagnosis and clinical therapy guidance.In the partâ… , by developing a new in situ tissue slide-based SELEX, using the paraffin tissue sections from breast infiltrating ductal carcinoma patients as targets, and the adjacent normal tissue sections from the same case as controls, one of the enriched ssDNA aptamers, BC15, was selected from a nucleic acid library. The confocal imaging results indicated that the target of BC15 is mainly located within the cancer cell nuclei. The nuclease treatment experiment revealed that the target of BC15 was presumably a non-nucleic acid substance, such as a DNA-binding protein. Through the streptavidin-biotin affinity purification assay followed by mass spectrometry identification and Western Blot confirmation, the target of BC15 was characterized to be hnRNP A1, which was further verified to be specifically and highly expressed in cancerous tissues of breast by hnRNP A1 antibody immunostaining as well as Western Blot. By studying numbers of clinical samples, BC15 aptamer was not only used to probe cancer cells in tissues from different pathological types of breast cancers but also used to probe many kinds of tumor including endometrial carcinoma, lung carcinoma, cervix carcinoma and liver carcinoma, etc. The aptamer was prone to bind the cancer tissues with low-medium degree of pathological differentiation and recognized the malignant cells in these tissues. BC15 can recognize the liver cancer samples those with high-medium degree of pathological differentiation from AFP-negative patients, indicating that combing use of BC15 with AFP may improve the clinical detection rate of liver cancer. Besides a direct use in monitoring of cancer cells, aptamer BC15 has a potential application in cancer therapy by competing with telomere DNA repeat sequence for hnRNP A1 binding which may result in shortening of telomeres and cell death. Therefore, tissue slide-based SELEX holds a promise in identifying tumor markers and developing specific molecular probes for cancer diagnosis and therapy.In the partâ…¡, in order to let BC15 autonomously enter the cell to play its role, we performed live cell subtractive SELEX using 4T1luc as target and Balb/c 3T3 as subtractive target to obtain an aptamer as effective delivery vehicle for BC15 aptamer and other nucleic acid fragment into targeted cancer cells. After nine rounds of selection, the highly enriched oligonucleic acid pool was sequenced and then grouped under different families on the basis of the homology of primary sequence and the similarity of secondary structure. Four sequences from different families were picked out for further characterization by confocal imagining and flow cytometry analysis. One of the aptamer termed s17 was verified to have the ability of delivery small nucleic acid fragment into target cells, suggesting the targeting properties of aptamer also can be exploited for localizing therapeutic agents to tumors, hence reducing off-target effects or other unwanted side effects. This novel"escort aptamers"may provide a potential for in vivo diagnosis and therapy use as targeting agents.In the partâ…¢, due to the limitations of traditional selection methods, we aimed to develop a new way for rapid, efficient and micro-sample selection by introducing the capillary electrophoresis technology into the SELEX procedure. Using the GFP expressed cells as target and FITC labeled ssDNA as initial library, we used CE-LIF to perform the first round of selection, and the flow cytometric analysis results revealed that only by a round of selection, the specific aptamers have been enriched, indicating the feasibility of this method to evolve aptamers. The successful development of CE-CELL-SELEX will pave the way for detection the clinical micro-sample such as circulating tumor cells, and for the rapid and batched selection of tumor biomarkers.