Department of Laboratory Medicine, Children's Hospital of Shanxi Province, Taiyuan, China.
Department of Laboratory Medicine, Shanxi Provincial People's Hospital, Taiyuan, China.
J Clin Lab Anal. 2021 Apr;35(4):e23718. doi: 10.1002/jcla.23718. Epub 2021 Feb 1.
This study was to find the aptamers with high affinity and specificity binding to acute promyelocytic leukemia (APL) NB4 cell line. These aptamers targeted NB4 cells were selected from a random single-stranded DNA (ssDNA) library of systematic evolution of ligands by exponential enrichment (CELL-SELEX). The binding rate of FITC-ssDNA library and NB4 cells was monitored using flow cytometry and fluorescence microscope. After cloned and sequenced, the structure, specificity, and affinity of these candidate aptamers were further analyzed. After a total of 19 rounds of selection, the ssDNA library was enriched and the BR (19.9%) of the 16th round was 12 times of the first round (1.6%). Three enriched aptamers were obtained from 21 positive clones of the 16th round, and the predicted secondary structures of these aptamers were mainly stem-loop. The aptamer CX9 had the highest affinity, and the equilibrium dissociation constant (Kd) was 16.2 nM. The fluorescence intensity of CX9 binding to NB4 cells was stronger than HL60 and K562 cells under fluorescence microscopy. The study indicates that aptamer CX9 exhibits high affinity and specificity with NB4 cells and lay a foundation for the rapid diagnostic method to detect APL with fluorescence-labeled aptamer.
本研究旨在寻找与急性早幼粒细胞白血病（APL）NB4 细胞系具有高亲和力和特异性结合的适体。这些针对 NB4 细胞的适体是通过指数富集的配体系统进化（CELL-SELEX）从随机单链 DNA（ssDNA）文库中筛选出来的。使用流式细胞术和荧光显微镜监测 FITC-ssDNA 文库与 NB4 细胞的结合率。克隆和测序后，进一步分析这些候选适体的结构、特异性和亲和力。经过 19 轮筛选，ssDNA 文库得到了富集，第 16 轮的 BR（19.9%）是第 1 轮的 12 倍（1.6%）。从第 16 轮的 21 个阳性克隆中获得了 3 个富集的适体，这些适体的预测二级结构主要为茎环结构。适体 CX9 具有最高的亲和力，平衡解离常数（Kd）为 16.2 nM。荧光显微镜下，CX9 与 NB4 细胞的结合荧光强度强于 HL60 和 K562 细胞。该研究表明，适体 CX9 与 NB4 细胞具有高亲和力和特异性，为荧光标记适体快速诊断 APL 奠定了基础。
