Mansoureh Haghighi
Mahak Hospital, Iran
Title: Cell-SELEX selection and characterization of 2 single-stranded DNA aptamers binding human 3 B-cell surface protein CD20
Biography
Biography: Mansoureh Haghighi
Abstract
The B-lymphocyte antigen CD20 (CD20) is a target of active agents in the diagnostic and 12 treatment of all types of B-cell lymphomas and leukemia. The extracellular domain loops of the 13 CD20 protein could be suitable targets for single-stranded nucleic acid oligomer (aptamers). 14 a(ssDNA) aptamers binding CD20, and to characterize their potency. CD20 was expressed in the 15 human embryonic kidney cells (HEK293T) and ssDNA library containing 52 randomized 16 nucleotides (nt) flanked by two 18-nt primers sites was constructed. Elevenrounds of Cell-SELEX 17 were performed and the selected ssDNA pool was amplified by polymerase chain reaction(PCR), 18 ligated with pTZ57T/A –pcDAS3.1 Hygro(+) vector and used to transform competent Escherichia 19 coli TOP10 cells. The plasmid o resulted clones were extracted and sequenced by M13 forward 20 primers. The results of sequencing revealed 10 different aptamers that shared some common 21 conserved regions. The secondary structures of the aptamers were predicted using the DNAMAN 22 software and three aptamers with the highest thermodynamic stability were selected (AP-1 to 23 AP-3). AP-1 aptamer was the most thermodynamically stable (ΔGAP-1 = -10.87 kcal/mol) with a 24 highest binding affinity (96.91 ± 4.5 nM) to the membranous CD20 protein. Further studies demand 25 to develop methods to use these apatmaers for diagnostic and therapeutic purposes in 26 CD20-related diseases.