<abstract xmlns="http://www.w3.org/1999/xhtml">

<sec id="j_raon-2018-0014_s_005">
<h3>Background</h3>
<p>Detection of circulating lung cancer cells with cancer-stem like characteristics would represent an improved tool for disease prognosis. However, current antibodies based methods have some disadvantages and therefore cell SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was used to develop DNA aptamers, recognizing cell surface markers of non-small lung carcinoma (NSLC) cells.</p>
</sec>
<sec id="j_raon-2018-0014_s_006">
<h3>Materials and methods</h3>
<p>The human adenocarcinoma cell line A549 was used for selection in seven cell SELEX cycles. We used human blood leukocytes for negative selection, and lung stem cell protein marker CD90 antibody binding A549 cells for positive selection.</p>
</sec>
<sec id="j_raon-2018-0014_s_007">
<h3>Results</h3>
<p>The obtained oligonucleotide sequences after the seventh SELEX cycle were subjected to <italic>in silico</italic> selection analysis based on three independent types of bioinformatics approaches, selecting two closely related aptamer candidates in terms of consensus sequences, structural motifs, binding affinity (Kd) and stability (ΔG). We selected and identified the aptamer A155_18 with very good binding characteristics to A459 cells, selected for CD90 antibody binding. The calculated phylogenetic tree showed that aptamers A155_18 and the known A549 cell aptamer S6 have a close structural relationship. MEME sequence analysis showed that they share two unique motifs, not present in other sequences.</p>
</sec>
<sec id="j_raon-2018-0014_s_008">
<h3>Conclusions</h3>
<p>The novel aptamer A155_18 has strong binding affinity for A549 lung carcinoma cell line subpopulation that is expressing stem cell marker CD90, indicating a possible stemness, characteristic for the A459 line, or a subpopulation present within this cell line. This aptamer can be applied as diagnostic tool, identifying NSLC circulating cells.</p>
</sec>
</abstract>

Background
Detection of circulating lung cancer cells with cancer-stem like characteristics would represent an improved tool for disease prognosis. However, current antibodies based methods have some disadvantages and therefore cell SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was used to develop DNA aptamers, recognizing cell surface markers of non-small lung carcinoma (NSLC) cells.


Materials and methods
The human adenocarcinoma cell line A549 was used for selection in seven cell SELEX cycles. We used human blood leukocytes for negative selection, and lung stem cell protein marker CD90 antibody binding A549 cells for positive selection.


Results
The obtained oligonucleotide sequences after the seventh SELEX cycle were subjected to in silico selection analysis based on three independent types of bioinformatics approaches, selecting two closely related aptamer candidates in terms of consensus sequences, structural motifs, binding affinity (Kd) and stability (ΔG). We selected and identified the aptamer A155_18 with very good binding characteristics to A459 cells, selected for CD90 antibody binding. The calculated phylogenetic tree showed that aptamers A155_18 and the known A549 cell aptamer S6 have a close structural relationship. MEME sequence analysis showed that they share two unique motifs, not present in other sequences.


Conclusions
The novel aptamer A155_18 has strong binding affinity for A549 lung carcinoma cell line subpopulation that is expressing stem cell marker CD90, indicating a possible stemness, characteristic for the A459 line, or a subpopulation present within this cell line. This aptamer can be applied as diagnostic tool, identifying NSLC circulating cells.

<italic>In silico</italic> selection approach to develop DNA aptamers for a stem-like cell subpopulation of non-small lung cancer adenocarcinoma cell line A549

Department of Genetic Toxicology and Cancer Biology

Instrumentation and Process Control, Centre for Biotechnology

Radiology and Oncology

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

 selection approach to develop DNA aptamers for a stem-like cell subpopulation of non-small lung cancer adenocarcinoma cell line A549

Background
Detection of circulating lung cancer cells with cancer-stem like characteristics would represent an improved tool for disease prognosis. However,...

In silico selection approach to develop DNA aptamers for a stem-like cell subpopulation of non-small lung cancer adenocarcinoma cell line A549

Article Category: Research Article

Data publikacji: 25 mar 2018

Zakres stron: 152 - 159

Otrzymano: 24 lis 2017

Przyjęty: 26 lut 2018

DOI: https://doi.org/10.2478/raon-2018-0014

© 2018 Mateja Vidic, Tina Smuc, Nika Janez, Michael Blank, Tomaz Accetto, Jan Mavri, Isis C. Nascimento, Arthur A. Nery, Henning Ulrich, Tamara T. Lah, published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

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