| Erythropoietin (EPO) is a kind of significant occurring hematopoietic growth factor and produced mainly in the kidney, which has the ability of promoting the proliferation and differentiation of erythroid progenitor cells and enriches the count of oxygen-carrying red blood cells. In recent years, the technique of transfection of EPO gene in mammalian cell lines makes large scale production of biologically active recombinant human EPO (rHuEPO) convenient with the development of genetic engineering. In all kinds of EPO preparations, rHuEPO-αand rHuEPO-β, have been extensively used in clinic to treat for anemia associated with renal disease and cancer.It is possible for rHuEPO-αto be abused in endurance sports to improve the performance by many athletes for its pharmacological action of enhancing the count of oxygen-carrying red blood cells. As a kind of well-known doping peptide hormone, erythropoiesis-stimulating agents are labeled clearly in the prohibited list of World Anti-Doping Code. Immunological or mass spectrometric-based techniques are mainly employed for its analysis with the aid of antibodies as a sensing and enrichment element. However, the limitation of anti-EPO antibody, such as batch-to-batch variability, immunogenicity and thermolability, will hinder the improvement of specificity and sensitivity in immunoassays. The effectively analytical strategies are greatly needed for detection of rHuEPO with the development of new recognition element.Aptamers are single-stranded DNA, RNA, or modified nucleic acids that have the ability to form defined tertiary structures for specific target binding. They have the similar recognition characterization as antibody and have no immunogenicity, easy preparation by chemical synthesis in vitro and strong thermal stability, these unique merits of aptamer are providing a promising alternative way to pursue affinity ligands with high affinity and high specificity for rHuEPO-α. So our group adopted agglutinin-directed Systematic Evolution of Ligands by EXponential enrichment (SELEX) technique to generate aptamers for rHuEPO-α, and a series of aptamers with specifity for rHuEPO-a were successfully obtained from 80nt ssDNA library after 8 selection rounds. Some aptamers with high affinity and specificity were chosen to carry out structure analysis. Finally, a new pattern of aptameric molecular beacon strategy was developed for the detection of rHuEPO-a basd on aptamer 807-39nt with naturally stem-loop secondary structure.The dissertation contains four parts, the first part is an overview on three aspects including instruction of function and purpose of EPO, development of SELEX technology and principle of molecular beacon. Total 115 references were cited.In the second part, the selection procedure of aptamer with high affinity and specificity for rHuEPO-a were introduced in detail. The wheat germ agglutinin (WGA)-directed affinity chromatographic SELEX was adopted to bind the sialic acids moiety of rHuEPO-a, and exposing peptide chains for selection, then a high-concentrated N-acetylglucosamine solution was applied to elute the complex of rHuEPO-a and ssDNAs by the displacement of sialic acids moiety from WGA-Sepharose. After 8 rounds of selection, the enrichment efficiency of oligonucleotides was obviously improved from 13.8% to 64.0% by determination of the percentage of bound ssDNAs and the selectivity of ssDNAs for rHuEPO-a was increased from 1.4 to 10.6. The clones sequenced from the 8th pool were divided into four families, the characteristics of these secondary structure present tight hairpins, multi-branched hairpins and few or no hairpins. The dissociation constants of some sequences were nM level, the sequence 807 can bind with rHuEPO-a with the highest affinity and specificity (Kd=82±32 nM).The third part is mainly on the truncation research of aptamers for rHuEPO-a. The sequences 807 and 813 with relative lower Gibbs’ energy and nanomole dissociation constant were selected to carry out truncation research in order to optimize and find the effective binding motif. The random sequence 807-39nt with tight hairpin structure truncated from the most frequently repeated sequence 807 exhibited by far the highest binding for rHuEPO-a, which can account for the superior binding capacity of the hairpin structure. The aptamer 813-42nt was the shortest motif truncated from 813 for binding with rHuEPO-a, EMS A results indicate that the stem forming between the region sequence and partly 3’primer region might facilitate the binding capacity.The secondary structure of 807-39nt is composed of a G-rich loop and a 5bp stem. A series of stem and loop variation experiments show that the recognition is occurred mainly in the loop part and the stem is functioned as a support to fix an enclosed loop in order to stabilize the complex of rHuEPO-α. Also the binding site of target rHuEPO-αwas further determined by the deglycosylation experiment, rHuEPO-αwithout N-glycosylation still remain the binding ability with 807-39nt. We might speculate that the binding site of protein is mainly the amino acid sequence, which is further confirmed by the binding between 807-39nt and Injection of Yi Bi Ao or Recormn. Also the results of immunohistochemistry experiments on normal urothelium showed that the specificity of aptamer 807-39nt was superior than anti-rHuEPO-αmAb AE7A5, and the aptamer 807-39nt has the ability to bind with malignant urothelium.In the fourth part, we designed the aptameric molecular beacon (MB) for rHuEPO-αdetection, based on the stem-loop secondary structure of 807-39nt. Both "Signal-on" and "Signal-off’ MB modes were developed, respectively, in which the conformational alteration of aptamer before and after binding to rHuEPO-a can be demonstrated in terms of the correspondingly fluorescent changes. Systematic optimization of parameters in "Signal-on" mode were carried out, the choice of QDNA length, the hybridization site of a small supplementary DNA (SDNA) stabilizer, and the existence of Mg2+cation played essential roles for the high sensitivity. With the optimized conditions, a convenient and sensitive determination of 1 nM rHuEPO-αwithout any preconcentration was achieved with the LOD of 0.4 nM.In summary, we successfully developed the agglutinin-directed affinity chromatographic SELEX to obtain a series of ssDNA aptamers with high affinity and specificity for rHuEPO-α. The structure and function researches were carried out with aptamer 807 and 813, the chief binding sites were determined preliminaryly. The best aptamer 807-39nt with Kd value of 39±27 nM proposed here can be set as powerful biosensors to facilitate new methods for rHuEPO-αand develop effective diagnostic reagent for rHuEPO-αas an alternative approach to antibody. |
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