Selection and Characterization of DNA Aptamers Targeting hLCN6 Protein for Sperm Capture

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. In this project, we developed a DNA aptamer-based system for sperm separation and purification from mixed stain samples by targeting sperm surface proteins. Human lipocalin 6 (hLCN6) is an epididymal secreted protein that binds to the head and tail of sperm cells and associated with sperm maturation. Using systematic evolution of ligands by exponential enrichment (SELEX) technology, aptamers that bind with high affinity and specificity to hLCN6 were screened from a random single-stranded DNA (ssDNA) library using magnetic bead-bound hLCN6 as target. The enriched library was obtained after 15 SELEX rounds. Of hLCN6-binding aptamer variants, 19 were further classified into one of the four groups based on their N60 random sequence regions, wherein one representative from each group was characterized. Prediction analysis of the secondary structure suggested discrete features with typical loop and stem motifs. Binding capability of selected aptamers was investigated by quantitative PCR, and aptamer H2 was found to be the most specific aptamer to sperm cells. The dissociation constant (K_d) of H2 aptamer was calculated as 3.21 ± 0.75 nM. Furthermore, H2 aptamer-coupled magnetic beads can recognize and capture sperm cells, which establishes the foundation of an approach for rapidly isolating sperm cells from mixed stains based on nucleic acid-protein interaction.