A novel hybrid approach to overcome defects of CE-SELEX and cell-SELEX in developing aptamers against aspartate β-hydroxylase
Background and purpose: Aptamers, a new category of molecular probes, are overthrowing antibodies in molecular diagnostics. However, there are serious problems with using aptamers for this application including poor or non-specific binding in vivo conditions. Systematic evolution of aptamers is achi...
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| Format: | Article |
| Language: | English |
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Wolters Kluwer Medknow Publications
2025-01-01
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| Series: | Research in Pharmaceutical Sciences |
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| Online Access: | https://journals.lww.com/10.4103/RPS.RPS_134_23 |
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| author | Hadi Bakhtiari Hamed Naghoosi Sina Sattari Mahmoud Vahidi Mehdi Shakouri Khomartash Ali Faridfar Mohsen Rajaeinejad Mohsen Nikandish |
| author_facet | Hadi Bakhtiari Hamed Naghoosi Sina Sattari Mahmoud Vahidi Mehdi Shakouri Khomartash Ali Faridfar Mohsen Rajaeinejad Mohsen Nikandish |
| author_sort | Hadi Bakhtiari |
| collection | DOAJ |
| description | Background and purpose:
Aptamers, a new category of molecular probes, are overthrowing antibodies in molecular diagnostics. However, there are serious problems with using aptamers for this application including poor or non-specific binding in vivo conditions. Systematic evolution of aptamers is achieved through various approaches including CE-SELEX and Cell-SELEX, each suffering its inevitable weaknesses. The shortcomings of negative selection and the lengthy procedure are Cell-SELEX’s main problems, while CE-SELEX is deprived of native targets. Here, we introduced a kind of hybrid CE-Cell-SELEX, named CEC hybrid-SELEX, for addressing these limitations in creating aptamer probes detecting human aspartate β-hydroxylase (ASPH), which is a well-established tumor biomarker, in cancer diagnostic investigations.
Experimental approach:
In our approach, the selected oligomer pool from the last cycle of CE-SELEX was sequenced and then subjected to 3 additional rounds of Cell-SELEX which provides native ASPH (CEC hybrid-SELEX). High-throughput sequencing was applied to achieve a comprehensive sight of the enriched pools. Further confirmatory investigations on oligomers with higher copy numbers were performed using flow cytometry.
Findings/Results:
Three selected oligomers, AP-CEC 1, AP-CEC 2, and AP-CEC 3, showing Kd values of 43.09 nM, 34.85 nM, and 35.92 nM, respectively, were achieved based on the affinity assessment of the ASPH-expressing cells.
Conclusion and implications:
Our research suggested that CEC hybrid-SELEX could help recognize which oligomers from CE-SELEX are more capable of binding native ASPH in vivo. |
| format | Article |
| id | doaj-art-5f18ec90675e44f5b3cf428162f8f0a1 |
| institution | DOAJ |
| issn | 1735-5362 1735-9414 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Wolters Kluwer Medknow Publications |
| record_format | Article |
| series | Research in Pharmaceutical Sciences |
| spelling | doaj-art-5f18ec90675e44f5b3cf428162f8f0a12025-08-20T03:02:10ZengWolters Kluwer Medknow PublicationsResearch in Pharmaceutical Sciences1735-53621735-94142025-01-01201657610.4103/RPS.RPS_134_23A novel hybrid approach to overcome defects of CE-SELEX and cell-SELEX in developing aptamers against aspartate β-hydroxylaseHadi BakhtiariHamed NaghoosiSina SattariMahmoud VahidiMehdi Shakouri KhomartashAli FaridfarMohsen RajaeinejadMohsen NikandishBackground and purpose: Aptamers, a new category of molecular probes, are overthrowing antibodies in molecular diagnostics. However, there are serious problems with using aptamers for this application including poor or non-specific binding in vivo conditions. Systematic evolution of aptamers is achieved through various approaches including CE-SELEX and Cell-SELEX, each suffering its inevitable weaknesses. The shortcomings of negative selection and the lengthy procedure are Cell-SELEX’s main problems, while CE-SELEX is deprived of native targets. Here, we introduced a kind of hybrid CE-Cell-SELEX, named CEC hybrid-SELEX, for addressing these limitations in creating aptamer probes detecting human aspartate β-hydroxylase (ASPH), which is a well-established tumor biomarker, in cancer diagnostic investigations. Experimental approach: In our approach, the selected oligomer pool from the last cycle of CE-SELEX was sequenced and then subjected to 3 additional rounds of Cell-SELEX which provides native ASPH (CEC hybrid-SELEX). High-throughput sequencing was applied to achieve a comprehensive sight of the enriched pools. Further confirmatory investigations on oligomers with higher copy numbers were performed using flow cytometry. Findings/Results: Three selected oligomers, AP-CEC 1, AP-CEC 2, and AP-CEC 3, showing Kd values of 43.09 nM, 34.85 nM, and 35.92 nM, respectively, were achieved based on the affinity assessment of the ASPH-expressing cells. Conclusion and implications: Our research suggested that CEC hybrid-SELEX could help recognize which oligomers from CE-SELEX are more capable of binding native ASPH in vivo.https://journals.lww.com/10.4103/RPS.RPS_134_23aptamersaspartate β-hydroxylasecapillary electrophoresishigh-throughput nucleotide sequencingselex aptamer technique |
| spellingShingle | Hadi Bakhtiari Hamed Naghoosi Sina Sattari Mahmoud Vahidi Mehdi Shakouri Khomartash Ali Faridfar Mohsen Rajaeinejad Mohsen Nikandish A novel hybrid approach to overcome defects of CE-SELEX and cell-SELEX in developing aptamers against aspartate β-hydroxylase Research in Pharmaceutical Sciences aptamers aspartate β-hydroxylase capillary electrophoresis high-throughput nucleotide sequencing selex aptamer technique |
| title | A novel hybrid approach to overcome defects of CE-SELEX and cell-SELEX in developing aptamers against aspartate β-hydroxylase |
| title_full | A novel hybrid approach to overcome defects of CE-SELEX and cell-SELEX in developing aptamers against aspartate β-hydroxylase |
| title_fullStr | A novel hybrid approach to overcome defects of CE-SELEX and cell-SELEX in developing aptamers against aspartate β-hydroxylase |
| title_full_unstemmed | A novel hybrid approach to overcome defects of CE-SELEX and cell-SELEX in developing aptamers against aspartate β-hydroxylase |
| title_short | A novel hybrid approach to overcome defects of CE-SELEX and cell-SELEX in developing aptamers against aspartate β-hydroxylase |
| title_sort | novel hybrid approach to overcome defects of ce selex and cell selex in developing aptamers against aspartate β hydroxylase |
| topic | aptamers aspartate β-hydroxylase capillary electrophoresis high-throughput nucleotide sequencing selex aptamer technique |
| url | https://journals.lww.com/10.4103/RPS.RPS_134_23 |
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