Selecting Of Aptamers Binding To CD33~+/CD34~- Cells From The Patients With Acute Myeloblastic Leukemia Of M2 Subtype And Their Structure Analysis

Background and Objective:The diagnosis and classification of acute myeloblastic leukemia (AML) are currently based mainly on the MICM typing method incorporated Morphology, Immunology, Cytogenetics and Molecular Biology. However, this method is still to be improved since the mechanism of leukogenesis and development of AML especially the specific molecular marker of leukemia cells is not yet clear. In recent years, with the development and improvement of systematic evolution of ligands by exponential enrichment (SELEX), selection of aptamer which can bind specific tumor cells, and can be regarded as specific molecular markers for cancer diagnosis, has received increasing attention. But, the specific aptamers of leukemia cells are still known little. Therefore, in this study cell-SELEX(C-SELEX) technology was applied to select aptamers specifically recognizing CD33+/CD34- cells from the patients with AML-M2 aptamer, this study can provide the basis for aptamer molecular diagnosis of AMLMethods:Firstly, single strand deoxyribonucleic acid (ssDNA) library was synthesize, in which contains a known sequence at both ends and a random sequence at the middle part in the deoxyribonucleic acid. Subsequently, leukemia CD33+/CD34- cells from the patients with AML-M2 was taken as targeted cells, normal CD33+/CD34- cells was taken as anti-selecting cells, the aptamers in the ssDNA library were then selected repeatedly by C-SELEX technology, and amplified by polymerase chain reaction (PCR) to generate sub-ssDNA library. During the experiment, PCR with fluorescently labeled primer and flow cytometry were used to analyze the aptamer's enrichment in sub-library. Finally, the final round product of the sub-ssDNA library was cloned. After sequencing, the primary and secondary structure of the aptamers were analyzed.Results:Agarose gel electrophoresis results, for each round PCR products, showed clear DNA bands. After 13 rounds of selection, the fluorescence intensity of the sub-ssDNA library binding to targeted cells ranged from 2.14% to 51.12%, and tended to saturation, reaching a steady state at 13 rounds. After cloning,30 clones were selected and sequenced. The sequence results showed that the aptamers contain one kind of the four conserved sequences of AAGTA, TATCT, AGATG and AAATT in their primary structure, but eight aptamers contain no any one kind of the conserved sequence. Secondary structure analysis indicated that four stem-loop and loop simulation convex structure are existed in the aptamers.Conclusion:In this study, aptamers specific binding to CD33+/CD34- cells from the patients of AML-M2 subtype are successful selected by C-SELEX technology, providing the basis for the further study of the aptamer properties and the molecular diagnosis of leukemia.