title: Hybrid Approach Combining Boronate Affinity Magnetic Nanoparticles and Capillary Electrophoresis for Efficient Selection of Glycoprotein-Binding Aptamers.
abstract: Capillary electrophoresis (CE) and magnetic beads have been widely used for the selection of aptamers owing to their efficient separation ability. However, these methods alone are associated with some apparent drawbacks. CE suffers from small injection volumes and thereby only a limited amount of aptamer can be collected at each round. While the magnetic beads approach is often associated with tedious procedure and nonspecific binding. Herein we present a hybrid approach that combines the above two classical aptamer selection methods to overcome the drawbacks associated with these methods alone. In this hybrid method, one single round selection by boronate affinity magnetic nanoparticles (BA-MNPs) was first performed and then followed by a CE selection of a few rounds. The BA-MNPs-based selection eliminated nonbinding sequences, enriching effective sequences in the nucleic acid library. While the CE selection, which was carried out in free solutions, eliminated steric hindrance effects in subsequent selection. Two typical glycoproteins, Ribonuclease B (RNase B) and alkaline phosphatase (ALP), were used as targets. This hybrid method allowed for efficient selection of glycoprotein-binding aptamers within 4 rounds (1 round of BA-MNPs-based selection and 3 rounds of CE selection) and the dissociation constants reached 10-8M level. The hybrid selection approach exhibited several significant advantages, including speed, affinity, specificity, and avoiding negative selection. Using one of the selected ALP-binding aptamers as an affinity ligand, feasibility for real application of the selected aptamers was demonstrated through constructing an improved enzyme activity assay.
authors: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University , Nanjing 210023, China.
authors: Xinglin
authors: X
authors: Li
authors: Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University, Clinical Cancer Institute of Nanjing University , 321 Zhongshan Road, Nanjing 210008, China.
authors: Yunjie
authors: Y
authors: He
authors: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University , Nanjing 210023, China.
authors: Yanyan
authors: Y
authors: Ma
authors: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University , Nanjing 210023, China.
authors: Zijun
authors: Z
authors: Bie
authors: Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University, Clinical Cancer Institute of Nanjing University , 321 Zhongshan Road, Nanjing 210008, China.
authors: Baorui
authors: B
authors: Liu
authors: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University , Nanjing 210023, China.
authors: Zhen
authors: Z
authors: Liu
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001665:Binding Sites; D001897:Boronic Acids / Q000737:chemistry*; D002417:Cattle; D019075:Electrophoresis, Capillary; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D012260:Ribonucleases / Q000032:analysis* / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D001897:Boronic Acids; D058185:Magnetite Nanoparticles; D012260:Ribonucleases; C029600:ribonuclease B; D000469:Alkaline Phosphatase