In vitro lectin-mediated selection and characterization of rHuEPO-α-binding ssDNA aptamers.
A lectin-mediated affinity chromatographic SELEX technique was developed to generate functional ssDNA aptamers for recombinant human erythropoietin-α (rHuEPO-α), an important pharmaceutical glycoprotein for the first time. Secondary structure analysis of the aptamer clones from sequential 6th, 7th, and 8th rounds showed that a certain fragment 'CGAGAT' in the 3' primer region could be used to trace increasing evolution stringency from its hybridization at different locations, in which a specific hybridization with its complement in the 5' primer region (aptamer 807) was evolved as the prevalent one with the simplest and most common motif. Characteristics of the aptamers with lower Gibbs' free energies and K(d) values (nM) were investigated. For aptamer 813, the minimer 813-42nt formed by the random and primer regions was indispensable for the specific binding with rHuEPO-α. While for aptamer 807, only the random region, that is, 807-39nt, was the functional motif. Further experiments of methylation, site-directed mutation and length variation showed that the loop of aptamer 807-39nt was the key region for binding with rHuEPO-α, and the stem should be considered as a stabilizing part. Lower cross-reactivity of aptamer 807-39nt was observed with human normal urothelium tissues than the anti-EPO monoclonal antibody AE7A5. Aptamer 807-39nt also exhibited a specific recognition for human bladder carcinoma cells and human urothelium tumors, which might provide a novel way to probe such tumors with overexpressed EPOs.
Zhang
Zhaoyang
Z
Beijing Institute of Pharmacology and Toxicology, No. 27 Taiping Road, Beijing 100850, China.
Guo
Lei
L
Guo
Aitao
A
Xu
Hua
H
Tang
Jijun
J
Guo
Xingjie
X
Xie
Jianwei
J
D000911:Antibodies, Monoclonal / Q000276:immunology; D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000302:isolation & purification; D004277:DNA, Single-Stranded / Q000737:chemistry; D004921:Erythropoietin / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D037102:Lectins / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins; D052156:SELEX Aptamer Technique; D013816:Thermodynamics; D001749:Urinary Bladder Neoplasms / Q000378:metabolism; D014571:Urologic Neoplasms / Q000378:metabolism
D000911:Antibodies, Monoclonal; D052157:Aptamers, Nucleotide; D004277:DNA, Single-Stranded; D037102:Lectins; D011994:Recombinant Proteins; D004921:Erythropoietin