A cocktail of structurally diverse single stranded DNA aptamers outperforms individual aptamers in sex sorting of bovine semen.

Aptamers are short, single-stranded nucleic acids that fold into three-dimensional scaffolds with diverse biological applications. Five Y-sperm specific ssDNA aptamers (Apt1-Apt5), selected from Systematic Evolution of Ligands by Exponential Enrichment with next-generation sequencing (SELEX-NGS) data based on read counts and minimum free energy (MFE), were evaluated for bovine semen sex sorting efficiency using droplet digital PCR (ddPCR) and PCR sexing of embryos generated by in vitro fertilization (IVF) using the unbound fraction of aptamer treated semen, which is expected to be enriched in X-bearing sperm following Y-sperm capture in the bound fraction. Apt1 yielded the highest X-sperm enrichment (64.3% by ddPCR; 69% by IVF) while a cocktail of all five aptamers further enhanced the enrichment (∼76% by ddPCR; ∼80% by IVF). NUPACK tube analysis revealed a positive association between dimerization propensity and X-sperm enrichment along with diverse multimeric structures in the cocktail tube. Additionally, aptamer cocktail exhibited negligible adverse effects on sperm functional and kinematic parameters. These findings indicate that multiple aptamers targeting a single cell type can achieve superior X-sperm enrichment and that dimer equilibrium concentration can refine aptamer selection from high-throughput data.