Abstract
ObjectiveThis study was conducted to generate single stranded DNA oligonucleotides with selective affinity to bovine spermatozoa, assess its binding potential and explore its potential utility in trapping spermatozoa from suspensions.MethodsA combinatorial library of 94 mer long oligonucleotide was used for systematic evolution of ligands by exponential enrichment (SELEX) with bovine spermatozoa. The amplicons from sixth and seventh rounds of SELEX were sequenced, and the reads were clustered employing cluster database at high identity with tolerance (CD-HIT) and FASTAptamer. The enriched nucleotides were predicted for secondary structures by Mfold, motifs by Multiple Em for Motif Elicitation and 5′ labelled with biotin/6-FAM to determine the binding potential and binding pattern.ResultsWe generated 14.1 and 17.7 million reads from sixth and seventh rounds of SELEX respectively to bovine spermatozoa. The CD-HIT clustered 78,098 and 21,196 reads in the top ten clusters and FASTAptamer identified 2,195 and 4,405 unique sequences in the top three clusters from the sixth and seventh rounds, respectively. The identified oligonucleotides formed secondary structures with delta G values between −1.17 to −26.18 kcal/mol indicating varied stability. Confocal imaging with the oligonucleotides from the seventh round revealed different patterns of binding to bovine spermatozoa (fluorescence of the whole head, spot of fluorescence in head and mid- piece and tail). Use of a 5′-biotin tagged oligonucleotide from the sixth round at 100 pmol with 4×106 spermatozoa could trap almost 80% from the suspension.ConclusionThe binding patterns and ability of the identified oligonucleotides confirms successful optimization of the SELEX process and generation of aptamers to bovine spermatozoa. These oligonucleotides provide a quick approach for selective capture of spermatozoa from complex samples. Future SELEX rounds with X- or Y- enriched sperm suspension will be used to generate oligonucleotides that bind to spermatozoa of a specific sex type.
Highlights
The growing demand of food for the human population that is predicted to reach ten billion by 2050 necessitates the use of modern biotechnology tools for sustainable production from both animal and agricultural resources
The concentration of the single-strand deoxyribonucleic acid (ssDNA) increased with the increase in AptaF concentration and the ratio of 10:1 which resulted in satisfactory amplification of ssDNA was selected to optimize the number of polymerase chain reaction (PCR) cycles and the template concentration
Molecules with greater binding affinity to targets generated through the process of systematic evolution of ligands by exponential enrichment (SELEX), from huge combinatorial oligonucleotide libraries are called aptamers and they have emerged as potential tool for application in different fields
Summary
The growing demand of food for the human population that is predicted to reach ten billion by 2050 necessitates the use of modern biotechnology tools for sustainable production from both animal and agricultural resources. Aptamers are alternative equivalents to antibodies that are generated by the process of systematic evolution of ligands by exponential enrichment (SELEX) employing high affinity pools of variant sequences of nucleic acid ligands for proteins or other immunological/ non-immunological structures and amplification of the bound species [6,7]. These short oligonucleotides are shown to have great potential due to their small size, high specificities, and their potential ability to differentiate between splice variants and post transcriptional modification of the same protein [8]. A successful attempt in this approach might provide an optimized protocol that could be applied for generation of alternate molecules with other specificities to bovine spermatozoa with potential downstream applications
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