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Asian-Australas J Anim Sci > Accepted Articles
DOI: https://doi.org/10.5713/ajas.20.0235    [Accepted] Published online August 21, 2020.
Generation of single stranded DNA with selective affinity to bovine spermatozoa
Sivadasan Pathiyil Vinod1  , Rajamani Vignesh1  , Mani Priyanka1  , Krishnaswamy Gopalan Tirumurugaan1,*  , Salem Nagalingam Sivaselvam2  , Gopal Dhinakar Raj3 
1Dept. of Animal Biotechnology Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai, India
2Dept of Animal Genetics and Breeding, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai, India
3Director, Centre for Animal Health Studies, Tamil Nadu Veterinary and Animal Sciences University, Chennai, India
Correspondence:  Krishnaswamy Gopalan Tirumurugaan,Email: kgtanuvas@gmail.com
Received: 15 April 2020   • Revised: 11 May 2020   • Accepted: 23 July 2020
Abstract
Objective
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
Methods
A 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 (MEME) and 5’ labelled with biotin/ 6-FAM to determine the binding potential and binding pattern
Results
We generated 14.1 and 17.7 million reads from sixth and seventh rounds of SELEX respectively to bovine spermatozoa. The CD-HIT clustered 78098 and 21196 reads in the top ten clusters and FASTAptamer identified 2195 and 4405 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 x 106 spermatozoa could trap almost 80% from the suspension.
Conclusion
The 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.
Keywords: Aptamers; Selective Enrichment; Binding; Surface Characteristics


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