Screening and Identification of Nucleic Acid Aptamers Specifically Binding to Human Lipocalin 6
CHEN Jiong1)*, ZHU Wei-Hao1), ZHAO Yue2)
1)Department of Forensic Biology, Henan University of Science and Technology, Luoyang 471023, Henan, China; 2)CITIC Heavy Industries Co, Luoyang 471003, Henan, China
Abstract：It is an urgent and difficult task to establish a simple and efficient method for identifying and isolating sperm cells from mixed stains in forensic science. Nucleic acid aptamers targeting sperm cell-surface proteins can be used for the separation and purification of sperms from mixed stain samples. Human lipocalin 6 (hLCN6) is an epididymal secreted protein that binds to the head and tail of sperm cells and is associated with sperm maturation. Using the systematic evolution of ligands by the exponential enrichment (SELEX) technique, magnetic bead-bound hLCN6 was used as the target molecule to screen for aptamers with high affinity and specificity to hLCN6 from a random single-stranded DNA (ssDNA) library.Through 15 rounds of positive selection and 3 rounds of negative selection, 24 clones were selected and subjected to sequence analysis. Subsequently, 4 candidate aptamers were selected and further examined for their binding affinity and specificity by enzyme-linked oligonucleotide adsorption (ELONA) and cell binding assays. One aptamer (H2) against hLCN6 with a high affinity and specificity was isolated and investigated by dot blotting and immunofluorescence staining. The result revealed that the candidate aptamer H2 with a dissociation constant of (3.21 ± 0.75) nmol/L was able to recognize and specifically bind to hLCN6. The aptamer H2 also showed high affinity and specificity to human sperms in vitro, which establishes the foundation for the separation of sperm cells from mixed stain based on nucleic acid-protein interactions and provides a new scheme.
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