The identification of single strand DNA aptamers which specifically bind to platelets using cell-SELEX technique
Aptamers are oligonucleotides that can be easily synthesized and bind to their targets with high affinity and specificity. Several aptamers specific to soluble factors of coagulation cascade have been produced, however, aptamers specific to platelet cell membrane molecules have not been reported yet...
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| Format: | Article |
| Language: | English |
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Ferdowsi University of Mashhad
2021-12-01
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| Series: | The Iranian Journal of Veterinary Science and Technology |
| Subjects: | |
| Online Access: | https://ijvst.um.ac.ir/article_41409_29aa90fed4fa4ecca7d05b756ef43b8b.pdf |
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| author | Fatemeh Alemi Mojtaba Sankian Alireza Haghparast Mohammad Reza Bassami Gholamreza Hashemi Tabar |
| author_facet | Fatemeh Alemi Mojtaba Sankian Alireza Haghparast Mohammad Reza Bassami Gholamreza Hashemi Tabar |
| author_sort | Fatemeh Alemi |
| collection | DOAJ |
| description | Aptamers are oligonucleotides that can be easily synthesized and bind to their targets with high affinity and specificity. Several aptamers specific to soluble factors of coagulation cascade have been produced, however, aptamers specific to platelet cell membrane molecules have not been reported yet. We aimed to discover DNA aptamers that specifically bind to human platelets. The cell-SELEX method was used for aptamer discovery. Synthetic 79 nucleotides length single-strand oligonucleotides were used as a library. Ultra-pure platelets were prepared using differential centrifugation steps and magnetic-bead-assisted removal of contaminating cells. The FITC-labeled forward primer was used for amplification of the selected oligonucleotides by PCR, and Lambda exonuclease was used for digestion of the lagging strand. After 12 rounds of cell-SELEX, selected oligos were amplified and cloned to pTG19-T vector, transfected into E. coli (TOP10) and sequenced. Sequences of aptamers from 200 individual positive colonies were aligned and seven clusters were identified. Representative aptamers were amplified and their affinity, specificity, and digestibility of their targets were evaluated. Interferences of the aptamers to two platelet function tests were also investigated. Affinity (KD) of the representative aptamers were between 109 and 340 nM. Trypsin exposure of the platelets completely abolished the binding of the 7 aptamers to the targets. The binding of the four aptamers fully protected their target molecules from digestion. No one of the aptamers changed the parameters of the platelet function tests. Seven aptamers specific to platelets were identified and characterized. These aptamers may have potentially diverse applications in the diagnosis or treatment of platelet disorders. |
| first_indexed | 2024-12-10T19:31:35Z |
| format | Article |
| id | doaj.art-65152fd7a805483ea5de091d3076a52c |
| institution | Directory Open Access Journal |
| issn | 2008-465X 2423-6306 |
| language | English |
| last_indexed | 2024-12-10T19:31:35Z |
| publishDate | 2021-12-01 |
| publisher | Ferdowsi University of Mashhad |
| record_format | Article |
| series | The Iranian Journal of Veterinary Science and Technology |
| spelling | doaj.art-65152fd7a805483ea5de091d3076a52c2022-12-22T01:36:14ZengFerdowsi University of MashhadThe Iranian Journal of Veterinary Science and Technology2008-465X2423-63062021-12-01132688310.22067/ijvst.2021.72518.107941409The identification of single strand DNA aptamers which specifically bind to platelets using cell-SELEX techniqueFatemeh Alemi0Mojtaba Sankian1Alireza Haghparast2Mohammad Reza Bassami3Gholamreza Hashemi Tabar4Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.Immunology Research Center, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran & Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran.Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran & Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran.Aptamers are oligonucleotides that can be easily synthesized and bind to their targets with high affinity and specificity. Several aptamers specific to soluble factors of coagulation cascade have been produced, however, aptamers specific to platelet cell membrane molecules have not been reported yet. We aimed to discover DNA aptamers that specifically bind to human platelets. The cell-SELEX method was used for aptamer discovery. Synthetic 79 nucleotides length single-strand oligonucleotides were used as a library. Ultra-pure platelets were prepared using differential centrifugation steps and magnetic-bead-assisted removal of contaminating cells. The FITC-labeled forward primer was used for amplification of the selected oligonucleotides by PCR, and Lambda exonuclease was used for digestion of the lagging strand. After 12 rounds of cell-SELEX, selected oligos were amplified and cloned to pTG19-T vector, transfected into E. coli (TOP10) and sequenced. Sequences of aptamers from 200 individual positive colonies were aligned and seven clusters were identified. Representative aptamers were amplified and their affinity, specificity, and digestibility of their targets were evaluated. Interferences of the aptamers to two platelet function tests were also investigated. Affinity (KD) of the representative aptamers were between 109 and 340 nM. Trypsin exposure of the platelets completely abolished the binding of the 7 aptamers to the targets. The binding of the four aptamers fully protected their target molecules from digestion. No one of the aptamers changed the parameters of the platelet function tests. Seven aptamers specific to platelets were identified and characterized. These aptamers may have potentially diverse applications in the diagnosis or treatment of platelet disorders.https://ijvst.um.ac.ir/article_41409_29aa90fed4fa4ecca7d05b756ef43b8b.pdfcell-selexplateletdna aptamerplatelet-specific aptamer |
| spellingShingle | Fatemeh Alemi Mojtaba Sankian Alireza Haghparast Mohammad Reza Bassami Gholamreza Hashemi Tabar The identification of single strand DNA aptamers which specifically bind to platelets using cell-SELEX technique The Iranian Journal of Veterinary Science and Technology cell-selex platelet dna aptamer platelet-specific aptamer |
| title | The identification of single strand DNA aptamers which specifically bind to platelets using cell-SELEX technique |
| title_full | The identification of single strand DNA aptamers which specifically bind to platelets using cell-SELEX technique |
| title_fullStr | The identification of single strand DNA aptamers which specifically bind to platelets using cell-SELEX technique |
| title_full_unstemmed | The identification of single strand DNA aptamers which specifically bind to platelets using cell-SELEX technique |
| title_short | The identification of single strand DNA aptamers which specifically bind to platelets using cell-SELEX technique |
| title_sort | identification of single strand dna aptamers which specifically bind to platelets using cell selex technique |
| topic | cell-selex platelet dna aptamer platelet-specific aptamer |
| url | https://ijvst.um.ac.ir/article_41409_29aa90fed4fa4ecca7d05b756ef43b8b.pdf |
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