Volume 48, Issue 3 (12-2024)
Research in Medicine 2024, 48(3): 9-20 |
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Ethics code: IR.SBMU.MSP.REC.1401.483
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Bahari M, Kargar Kheirabad A, Yeganeh F. Construction of a Chemically- Conjugated DNA Aptamer- Protein Hybrid Molecule to Develop the Enzyme- Linked Apta- Sorbent Assay. Research in Medicine 2024; 48 (3) :9-20
URL: http://pejouhesh.sbmu.ac.ir/article-1-3389-en.html
URL: http://pejouhesh.sbmu.ac.ir/article-1-3389-en.html
Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. , fyeganeh@sbmu.ac.ir
Abstract: (286 Views)
Background and Aim: Aptamers are single-stranded nucleic acid molecules, either DNA or RNA, selected for their high affinity and specificity to target molecules and advantages over antibodies. The current study aimed to establish a robust protocol for the chemical conjugation of an aptamer to horseradish peroxidase (HRP) enzyme using the heterobifunctional crosslinker sulfo- succinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate (sulfo- SMCC) to facilitate the development of aptamers in diagnostic methods. This method was used for aptamer conjugation for the first time.
Methods: The current study was performed in two steps: 1- Design and construction through an exploratory method and 2- Experimental investigation. This experimental study involved the activation of HRP with the sulfo- SMCC crosslinker. Post-activation, HRP was purified by size exclusion chromatography. The activated HRP was subsequently crosslinked with a thiolated aptamer via maleimide- thiol chemistry. The resulting HRP- aptamer conjugate was further purified using Amicon filters. A direct enzyme- linked aptasorbent assay (ELASA) was then performed to validate the conjugation efficiency and functionality of the aptamer- HRP complex. Statistical analyses were conducted using GraphPad Prism 10 software, applying ANOVA to assess significance.
Results: The research demonstrated that it is possible to make a protein- aptamer hybrid molecule using conjugation. During the conjugation process, the HRP enzyme was covalently linked to the NHS- ester end of the sulfo- SMCC linker through its amino group, resulting in stable amide bond formation. The thiolated aptamer was then conjugated to the linker via thioether linkage. To evaluate the efficacy of the conjugation, a direct ELASA assay was performed using varying concentrations of the HRP-aptamer conjugate (1, 2 and 5 μM) and each concentration was tested in duplicate. The data showed that different concentrations of the conjugated aptamer significantly affected the aptamer's performance. The average optical density (OD) for the positive control samples were 0.5, 1, 1.6, corresponding to increasing concentrations of the conjugate, while negative controls consistently showed an OD of 0.04. The statistical analysis confirmed that the differences in OD between positive and negative controls were significant (P < 0.05), demonstrating successful conjugation without losing aptamer functionality.
Conclusion: The findings of this investigation demonstrated that, initially, it is possible to generate a protein hybrid molecule, and that, secondly, its use is likely to be advantageous and it seems that conjugation of aptamer is possible without negatively affecting the performance of the aptamer.
Methods: The current study was performed in two steps: 1- Design and construction through an exploratory method and 2- Experimental investigation. This experimental study involved the activation of HRP with the sulfo- SMCC crosslinker. Post-activation, HRP was purified by size exclusion chromatography. The activated HRP was subsequently crosslinked with a thiolated aptamer via maleimide- thiol chemistry. The resulting HRP- aptamer conjugate was further purified using Amicon filters. A direct enzyme- linked aptasorbent assay (ELASA) was then performed to validate the conjugation efficiency and functionality of the aptamer- HRP complex. Statistical analyses were conducted using GraphPad Prism 10 software, applying ANOVA to assess significance.
Results: The research demonstrated that it is possible to make a protein- aptamer hybrid molecule using conjugation. During the conjugation process, the HRP enzyme was covalently linked to the NHS- ester end of the sulfo- SMCC linker through its amino group, resulting in stable amide bond formation. The thiolated aptamer was then conjugated to the linker via thioether linkage. To evaluate the efficacy of the conjugation, a direct ELASA assay was performed using varying concentrations of the HRP-aptamer conjugate (1, 2 and 5 μM) and each concentration was tested in duplicate. The data showed that different concentrations of the conjugated aptamer significantly affected the aptamer's performance. The average optical density (OD) for the positive control samples were 0.5, 1, 1.6, corresponding to increasing concentrations of the conjugate, while negative controls consistently showed an OD of 0.04. The statistical analysis confirmed that the differences in OD between positive and negative controls were significant (P < 0.05), demonstrating successful conjugation without losing aptamer functionality.
Conclusion: The findings of this investigation demonstrated that, initially, it is possible to generate a protein hybrid molecule, and that, secondly, its use is likely to be advantageous and it seems that conjugation of aptamer is possible without negatively affecting the performance of the aptamer.
Keywords: Aptamer, conjugation, crosslinkers, Enzyme- linked immunosorbent assay (ELISA), Enzyme- linked apta- sorbent assay (ELASA)
Type of Study: Original |
Subject:
Immunology
Received: 2024/06/18 | Accepted: 2024/11/6 | Published: 2025/01/20
Received: 2024/06/18 | Accepted: 2024/11/6 | Published: 2025/01/20
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