In vitro selection of single strand deoxyribonucleic acid aptamers binding to cells from patients with acute myeloblastic leukemia.
10.3969/j.issn.1672-7347.2012.08.003
- Author:
Ping ZHU
1
;
Guangping WANG
;
Shuqin ZHANG
;
Yajing XU
;
Minyuan PENG
;
Hui YIN
;
Yan CHEN
;
Sanqin TAN
;
Fangping CHEN
Author Information
1. Department of Hematology, Xiangya Hospital, Central South University, Changsha 410008, China.
- Publication Type:Journal Article
- MeSH:
Adolescent;
Adult;
Antigens, CD34;
genetics;
immunology;
Antigens, Differentiation, Myelomonocytic;
genetics;
immunology;
Aptamers, Nucleotide;
genetics;
metabolism;
DNA, Single-Stranded;
genetics;
Female;
Humans;
Leukemia, Myeloid, Acute;
genetics;
immunology;
Male;
Middle Aged;
SELEX Aptamer Technique;
Sialic Acid Binding Ig-like Lectin 3;
genetics;
immunology;
Young Adult
- From:
Journal of Central South University(Medical Sciences)
2012;37(8):771-776
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To screen aptamers binding CD33+/CD34- cells from patients with acute myeloblastic leukemia M2 subtype (AML-M2).
METHODS:CD33+/CD34- cells from patients with AML-M2 were taken as targeted cells, CD33+/ CD34- cells from normal people were taken as anti-selecting cells, and aptamers in the single strand deoxyribonucleic acid (ssDNA) library were then selected repeatedly by cell-systematic evolution of ligands by exponential enrichment (C-SELEX) technology, and amplified by polymerase chain reaction (PCR) to generate sub-ssDNA library. During the experiment, PCR amplification with fluorescently labeled primer and flow cytometry were performed to analyze the aptamers'enrichment of sub-library, and the final round product of the sub-ssDNA library was cloned. After the sequencing, the primary and secondary structures of the aptamers were analyzed.
RESULTS:Electrophoresis indicated that the product of PCR amplification for each round subssDNA library was able to see a clear DNA band in the agarose gel. After 13 rounds of screening, the fluorescence intensity of the sub-ssDNA library binding the cells ranged from 2.14% to 51.12%, reaching a steady state at the 13th round. A total of 30 clones were selected and sequenced, 22 of which contained 1 of the 4 conserved sequences of AAGTA, TATCT, AGATG and AAATT in their primary structure, but the remained eight aptamers contained none of the conserved sequence. Secondary structure analysis indicated that four stem-loops and loop simulation convex structures existed in the aptamers.
CONCLUSION:C-SELEX technology can be used to screen the aptamers binding primary cells from patients with leukemia. The aptamers selected from the CD33+/CD34- cells from the patients of AML-M2 subtype might be used for the diagnosis and treatment for leukemia.
