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A Novel ssDNA Aptamer Targeting Carcinoembryonic Antigen: Selection and Characterization
SIMPLE SUMMARY: Carcinoembryonic antigen (CEA), a well-known cancer biomarker that is used to diagnose various cancers, notably colorectal cancer, was used as a target to select and characterize single-stranded DNA aptamers. These short and compact nucleic acid sequences, known as aptamers, are beco...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9598387/ https://www.ncbi.nlm.nih.gov/pubmed/36290442 http://dx.doi.org/10.3390/biology11101540 |
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author | Yunussova, Nigara Sypabekova, Marzhan Zhumabekova, Zhazira Matkarimov, Bakhyt Kanayeva, Damira |
author_facet | Yunussova, Nigara Sypabekova, Marzhan Zhumabekova, Zhazira Matkarimov, Bakhyt Kanayeva, Damira |
author_sort | Yunussova, Nigara |
collection | PubMed |
description | SIMPLE SUMMARY: Carcinoembryonic antigen (CEA), a well-known cancer biomarker that is used to diagnose various cancers, notably colorectal cancer, was used as a target to select and characterize single-stranded DNA aptamers. These short and compact nucleic acid sequences, known as aptamers, are becoming ever more popular in both therapeutics and diagnostics. In this study, SELEX and NGS techniques were employed in the aptamer selection processes. Aptamer characterization techniques, such as enzyme-linked oligonucleotide assay (ELONA), dot blot, and bioinformatics assays, were conducted to observe the affinity binding of the selected aptamer to the target protein. Confocal microscopy results demonstrated how the fluorescently labelled aptamer sequence recognized CEA, which is expressed in HT-29 human colon adenocarcinoma cell line. These findings show that the selected aptamer has the potential to be employed as a recognition component in rapid detection systems, such as aptasensors. ABSTRACT: One of the major causes of a drastically shorter life expectancy and one of the most prevalent diseases in the world today is cancer. Given the data on the rise in cancer cases throughout the world, it is obvious that, despite the diagnostic techniques currently being used, there is a pressing need to develop precise and sensitive techniques for early diagnosis of the disease. A high degree of affinity and specificity towards particular targets is maintained by the short nucleic acid molecules known as aptamers. Aptamers outperform antibodies due to their unique benefits, such as their simplicity in synthesis and modification, lack of toxicity, and long-term stability. Utilizing an accurate recognition element and a robust signal transduction mechanism, molecular diagnostics can be extremely sensitive and specific. In this study, development of new single-stranded DNA aptamers against CEA for use in cancer diagnostics was accomplished using SELEX and NGS methods. As a result of 12 iterative SELEX rounds, nine aptamer candidates against CEA were developed. NGS comparative analysis revealed that round twelve had an enriched number of aptamers that were specifically bound, as opposed to round eight. Among the selected nine sequences characterized by bioinformatics analysis and ELONA, an aptamer sequence with the highest specificity and affinity for the target protein was identified and further examined. Aptamer sequence (6) was screened in a concentration-dependent assay, specificity analysis was performed, and its potential secondary and tertiary structures were predicted, which enabled us to test one of the possible putative interactions with CEA. Finally, aptamer sequence (6) labelled with a Cy5 fluorescent tag was used in confocal microscopy to observe its binding towards the CEA expressed in HT-29 human colon adenocarcinoma cell line. |
format | Online Article Text |
id | pubmed-9598387 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-95983872022-10-27 A Novel ssDNA Aptamer Targeting Carcinoembryonic Antigen: Selection and Characterization Yunussova, Nigara Sypabekova, Marzhan Zhumabekova, Zhazira Matkarimov, Bakhyt Kanayeva, Damira Biology (Basel) Article SIMPLE SUMMARY: Carcinoembryonic antigen (CEA), a well-known cancer biomarker that is used to diagnose various cancers, notably colorectal cancer, was used as a target to select and characterize single-stranded DNA aptamers. These short and compact nucleic acid sequences, known as aptamers, are becoming ever more popular in both therapeutics and diagnostics. In this study, SELEX and NGS techniques were employed in the aptamer selection processes. Aptamer characterization techniques, such as enzyme-linked oligonucleotide assay (ELONA), dot blot, and bioinformatics assays, were conducted to observe the affinity binding of the selected aptamer to the target protein. Confocal microscopy results demonstrated how the fluorescently labelled aptamer sequence recognized CEA, which is expressed in HT-29 human colon adenocarcinoma cell line. These findings show that the selected aptamer has the potential to be employed as a recognition component in rapid detection systems, such as aptasensors. ABSTRACT: One of the major causes of a drastically shorter life expectancy and one of the most prevalent diseases in the world today is cancer. Given the data on the rise in cancer cases throughout the world, it is obvious that, despite the diagnostic techniques currently being used, there is a pressing need to develop precise and sensitive techniques for early diagnosis of the disease. A high degree of affinity and specificity towards particular targets is maintained by the short nucleic acid molecules known as aptamers. Aptamers outperform antibodies due to their unique benefits, such as their simplicity in synthesis and modification, lack of toxicity, and long-term stability. Utilizing an accurate recognition element and a robust signal transduction mechanism, molecular diagnostics can be extremely sensitive and specific. In this study, development of new single-stranded DNA aptamers against CEA for use in cancer diagnostics was accomplished using SELEX and NGS methods. As a result of 12 iterative SELEX rounds, nine aptamer candidates against CEA were developed. NGS comparative analysis revealed that round twelve had an enriched number of aptamers that were specifically bound, as opposed to round eight. Among the selected nine sequences characterized by bioinformatics analysis and ELONA, an aptamer sequence with the highest specificity and affinity for the target protein was identified and further examined. Aptamer sequence (6) was screened in a concentration-dependent assay, specificity analysis was performed, and its potential secondary and tertiary structures were predicted, which enabled us to test one of the possible putative interactions with CEA. Finally, aptamer sequence (6) labelled with a Cy5 fluorescent tag was used in confocal microscopy to observe its binding towards the CEA expressed in HT-29 human colon adenocarcinoma cell line. MDPI 2022-10-20 /pmc/articles/PMC9598387/ /pubmed/36290442 http://dx.doi.org/10.3390/biology11101540 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Yunussova, Nigara Sypabekova, Marzhan Zhumabekova, Zhazira Matkarimov, Bakhyt Kanayeva, Damira A Novel ssDNA Aptamer Targeting Carcinoembryonic Antigen: Selection and Characterization |
title | A Novel ssDNA Aptamer Targeting Carcinoembryonic Antigen: Selection and Characterization |
title_full | A Novel ssDNA Aptamer Targeting Carcinoembryonic Antigen: Selection and Characterization |
title_fullStr | A Novel ssDNA Aptamer Targeting Carcinoembryonic Antigen: Selection and Characterization |
title_full_unstemmed | A Novel ssDNA Aptamer Targeting Carcinoembryonic Antigen: Selection and Characterization |
title_short | A Novel ssDNA Aptamer Targeting Carcinoembryonic Antigen: Selection and Characterization |
title_sort | novel ssdna aptamer targeting carcinoembryonic antigen: selection and characterization |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9598387/ https://www.ncbi.nlm.nih.gov/pubmed/36290442 http://dx.doi.org/10.3390/biology11101540 |
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