Screening And Cloning Of Aptamers Binding To The Surface Of CD33⁺/CD34⁺ Cells From The Patient With Acute Myeloid Leukemia Of M₂ Subtype And Their Sequences Analysis

Objective:Leukemia is a malignant disease derived from bone morrow hematopoietic stem cells. It is beneficial to treat leukemia and to improve the cure rate of leukemia if the leukemia can be exactly diagnosed. Systematic evolution of ligands by exponential enrichment (SELEX) is a new technology combined molecular biology and chemical composition, which can be used to screen aptamers binding specially to targeted cells, the latter is often used for the molecular diagnosis of cancer. Therefore, the aim of our study was to screen and obtain the high affinity aptamers binding to AML cells by the method of cell-systematic evolution of ligands by exponential enrichment (c-SELEX), which lay a foundation for diagnosis of leukemia.Methods:(cluster of differentiation antigen CD) CD33/CD34 cells were firstly positively sorted by the method of magnetic activated cell sorting (MACS) followed by the purity test. CD33+/CD34+cells from the acute myeloid leukemia(AML)-M2 patients were then acted as targeted cells, and normal CD33+/CD34+cells were acted as counter-targeted cells, the aptamers that specially bind to CD33+/CD34+cells from patients with AML-M2 were subsequently selected from the ssDNA library by the method of c-SELEX. The enrichment of the aptamers was analyzed. Finally, the aptamers obtained at the last round were amplified, cloned and sequenced, and each aptamer structure were further analyzed.Results:After 13 round of selection, the enrichment of aptamers in the random ssDNA library was ranged from 0.7% to 52.9%, and it reached steady state after 12th round selection. Sequence analysis for 30 aptamers showed that most of the aptamers bear one of the three conserved sequences of CCCCT, CTCTC, and CTCAC, But two of them carry not any one three conserved sequences, and the result of secondary structure analysis indicated that three different secondary structure were existed I these aptamers.Conclusions:In our study, the aptamers specially binding the CD33+/CD34+cells from patients with AML-M2 are successfully screened by the method of c-SELEX, which set a foundation for the further study on the molecular diagnosis of leukemia.