Animal Bioscience

Asian Australasian Association of Animal Production Societies (아세아태평양축산학회)
권호 표지
DOI QR코드

DOI QR Code

Generation of single stranded DNA with selective affinity to bovine spermatozoa

  • Vinod, Sivadasan Pathiyil (Department of Animal Biotechnology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University) ;
  • Vignesh, Rajamani (Department of Animal Biotechnology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University) ;
  • Priyanka, Mani (Department of Animal Biotechnology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University) ;
  • Tirumurugaan, Krishnaswamy Gopalan (Department of Animal Biotechnology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University) ;
  • Sivaselvam, Salem Nagalingam (Department of Animal Genetics and Breeding, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University) ;
  • Raj, Gopal Dhinakar (Centre for Animal Health Studies, Tamil Nadu Veterinary and Animal Sciences University)
  • Received : 2020.04.15
  • Accepted : 2020.07.23
  • Published : 2021.10.01
Download PDF
⟨ Previous Next ⟩

Abstract

Objective: This 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. 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 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 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. 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

Acknowledgement

Under funding from DBT, GOI, Govt. of India for the project BT/PR17667/AAQ/1/661 to the corresponding author (KG) and the facilities provided by Tamil Nadu Veterinary and Animal Sciences University, Chennai. The authors thank Dr. S. Ramesh, TANVUAS for his help in proof-reading the revised manuscript.

References

  1. Bradley MP. Immunological sexing of mammalian semen: current status and future options. J Dairy Sci 1989;72:3372-80. https://doi.org/10.3168/jds.S0022-0302(89)79500-X
  2. Blecher SR, Howie R, Li S, Detmar J, Blahut LM. A new approach to immunological sexing of sperm. Theriogenology 1999;52:1309-21. https://doi.org/10.1016/S0093-691X(99)00219-8
  3. Hafs HD, Kiddy CA. Sex ratio at birth - prospects for control; symposium. Champaign, IL, USA: American Society of Animal Science; 1971.
  4. van Munster EB, Stap J, Hoebe RA, te Meerman GJ, Aten JA. Difference in sperm head volume as a theoretical basis for sorting X- and Y- bearing spermatozoa: potentials and limitations. Theriogenology 1999;52:1281-93. https://doi.org/10.1016/S0093-691X(99)00217-4
  5. Johnson LA. Sex preselection by flow cytometric separation of X and Y chromosome-bearing sperm based on DNA difference: a review. Reprod Fertil Dev 1995;7:893-903. https://doi.org/10.1071/RD9950893
  6. Song KM, Lee S, Ban C. Aptamers and their biological applications. Sensors 2012;12:612-31. https://doi.org/10.3390/s120100612
  7. Ellington AD, Szostak JW. In vitro selection of RNA molecules that bind specific ligands. Nature 1990;346:818-22. https://doi.org/10.1038/346818a0
  8. Jayasena SD. Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clin Chem 1999;45:1628-50. https://doi.org/10.1093/clinchem/45.9.1628
  9. Pla-Roca M, Leulmi RF, Tourekhanova S, et al. Antibody colocalization microarray: a scalable technology for multiplex protein analysis in complex samples. Mol Cell Proteomics 2012;11:M111.011460. https://doi.org/10.1074/mcp.M111.011460
  10. Breaker RR. Are engineered proteins getting competition from RNA? Curr Opin Biotechnol 1996;7:442-8. https://doi. org/10.1016/S0958-1669(96)80122-4
  11. Nimjee SM, White RR, Becker RC, Sullenger BA. Aptamers as therapeutics. Annu Rev Pharmacol Toxicol 2017;57:61-79. https://doi.org/10.1146/annurev-pharmtox-010716-104558
  12. Amato T, Virgilio A, Pirone L, et al. Investigating the properties of TBA variants with twin thrombin binding domains. Sci Rep 2019;9:9184. https://doi.org/10.1038/s41598-019-45526-z
  13. Bahreyni A, Ramezani M, Alibolandi M, Hassanzadeh P, Abnous K, Taghdisi SM. High affinity of AS1411 toward copper; its application in a sensitive aptasensor for copper detection. Anal Biochem 2019;575:1-9. https://doi.org/10.1016/j.ab.2019.03.016
  14. Wilbanks B, Smestad J, Heider RM, Warrington AE, Rodriguez M, Maher LJ. Optimization of a 40-mer antimyelin DNA aptamer identifies a 20-mer with enhanced properties for potential multiple sclerosis therapy. Nucleic Acid Ther 2019;29:126-35. https://doi.org/10.1089/nat.2018.0776
  15. Wu Y, Midinov B, White RJ. Electrochemical aptamer-based sensor for real-time monitoring of insulin. ACS Sens 2019;4:498-503. https://doi.org/10.1021/acssensors.8b01573
  16. Soto Rodriguez PED, Nash VIC. Chapter 6-aptamer-based strategies for diagnostics. In: Filice M, Ruiz-Cabello J, editors. Nucleic acid nanotheranostics. Amsterdam, Netherlands: Elsevier; 2019. pp.189-211. https://doi.org/10.1016/B978-0-12-814470-1.00006-X
  17. Zhao H, Ma C, Chen M. A novel fluorometric method for inorganic pyrophosphatase detection based on G-quadruplex-thioflavin T. Mol Cell Probes 2019;43:29-33. https://doi.org/10.1016/j.mcp.2018.12.003
  18. Nimjee SM, Rusconi CP, Sullenger BA. Aptamers: an emerging class of therapeutics. Annu Rev Med 2005;56:555-83. https://doi.org/10.1146/annurev.med.56.062904.144915
  19. Platella C, Riccardi C, Montesarchio D, Roviello GN, Musumeci D. G-Quadruplex-based aptamers against protein targets in therapy and diagnostics. Biochim Biophys Acta Gen Subj 2017;1861:1429-47. https://doi.org/10.1016/j.bbagen.2016.11.027
  20. Campbell MA, Wengel J. Locked vs. unlocked nucleic acids (LNAvs.UNA): contrasting structures work towards common therapeutic goals. Chem Soc Rev 2011;40:5680-9. https://doi.org/10.1039/C1CS15048K
  21. Colley AJ, Buhr MM, Golovan SP. Selection of sex-specific aptamer probes to sperm. Theriogenology 2008;70:1384. https://doi.org/10.1016/j.theriogenology.2008.06.039
  22. Hoodbhoy T, Dean J. Insights into the molecular basis of sperm-egg recognition in mammals. Reproduction 2004;127:417-22. https://doi.org/10.1530/rep.1.00181
  23. Suri A. Sperm specific proteins-potential candidate molecules for fertility control. Reprod Biol Endocrinol 2004;2:10. https://doi.org/10.1186/1477-7827-2-10
  24. Lenzi A, Picardo M, Gandini L, Dondero F. Lipids of the sperm plasma membrane: from polyunsaturated fatty acids considered as markers of sperm function to possible scavenger therapy. Hum Reprod Update 1996;2:246-56. https://doi.org/10.1093/humupd/2.3.246
  25. Null AP, Hannis JC, Muddiman DC. Preparation of singlestranded PCR products for electrospray ionization mass spectrometry using the DNA repair enzyme lambda exonuclease. Analyst 2000;125:619-26. https://doi.org/10.1039/A908022H
  26. Velez TE, Singh J, Xiao Y, et al. Systematic evaluation of the dependence of deoxyribozyme catalysis on random region length. ACS Comb Sci 2012;14:680-7. https://doi.org/10.1021/co300111f
  27. McKeague M, McConnell EM, Toledo JC, et al. Analysis of in vitro aptamer selection parameters. J Mol Evol 2015;81:150-61. https://doi.org/10.1007/s00239-015-9708-6
  28. Silverman SK. Artificial functional nucleic acids: aptamers, ribozymes, and deoxyribozymes identified by in vitro selection. In: Li Y, Lu Y, editors. Functional nucleic acids for analytical applications. New York, NY, USA: Springer; 2009. pp. 47-108. https://doi.org/10.1007/978-0-387-73711-9_3
  29. Gyllensten UB, Erlich HA. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Natl Acad Sci USA 1988;85:7652-6. https://doi.org/10.1073/pnas.85.20.7652
  30. Higuchi RG, Ochman H. Production of single-stranded DNA templates by exonuclease digestion following the polymerase chain reaction. Nucleic Acids Res 1989;17:5865. https://doi.org/10.1093/nar/17.14.5865
  31. Williams KP, Bartel DP. PCR product with strands of unequal length. Nucleic Acids Res 1995;23:4220-1. https://doi.org/10.1093/nar/23.20.4220
  32. Cao X, Li S, Chen L, et al. Combining use of a panel of ssDNA aptamers in the detection of Staphylococcus aureus. Nucleic Acids Res 2009;37:4621-8. https://doi.org/10.1093/nar/gkp489
  33. Ward WS. Function of sperm chromatin structural elements in fertilization and development. Mol Hum Reprod 2010;16:30-6. https://doi.org/10.1093/molehr/gap080
  34. Daniels DA, Chen H, Hicke BJ, Swiderek KM, Gold L. A tenascin-C aptamer identified by tumor cell SELEX: systematic evolution of ligands by exponential enrichment. Proc Natl Acad Sci USA 2003;100:15416-21. https://doi.org/10.1073/pnas.2136683100
  35. Shangguan D, Li Y, Tang Z, et al. Aptamers evolved from live cells as effective molecular probes for cancer study. Proc Natl Acad Sci USA 2006;103:11838-43. https://doi.org/10.1073/pnas.0602615103
  36. Beier R, Boschke E, Labudde D. New strategies for evaluation and analysis of SELEX experiments. Biomed Res Int 2014;2014:849743. https://doi.org/10.1155/2014/849743
  37. Fu L, Niu B, Zhu Z, Wu S, Li W. CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics 2012;28:3150-2. https://doi.org/10.1093/bioinformatics/bts565
  38. Alam KK, Chang JL, Burke DH. FASTAptamer: a bioinformatic toolkit for high-throughput sequence analysis of combinatorial selections. Mol Ther Nucleic Acids 2015;4:e230. https://doi.org/10.1038/mtna.2015.4
  39. SantaLucia J. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci USA 1998;95:1460-5. https://doi.org/10.1073/pnas.95.4.1460
  40. Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 2003;31:3406-15. https://doi.org/10.1093/nar/gkg595
  41. Gansler J, Jaax M, Leiting S, et al. Structural requirements for the procoagulant activity of nucleic acids. PLoS One 2012;7:e50399. https://doi.org/10.1371/journal.pone.0050399
  42. Bailey TL, Williams N, Misleh C, Li WW. MEME: discovering and analyzing DNA and protein sequence motifs. Nucleic Acids Res 2006;34(Suppl 2):W369-73. https://doi.org/10.1093/nar/gkl198
  43. van der Meer DLM, Degenhardt T, Vaisanen S, et al. Profiling of promoter occupancy by PPARα in human hepatoma cells via ChIP-chip analysis. Nucleic Acids Res 2010;38:2839-50. https://doi.org/10.1093/nar/gkq012