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

Abstract

BackgroundDetection 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 methodsThe 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.ResultsThe 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.ConclusionsThe 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.

Selection of DNA Aptamers for Differentiation of Human Adipose-Derived Mesenchymal Stem Cells from Fibroblasts

“…Therefore, Apta 99 is the candidate aptamer with the greatest thermodynamic stability. Among the Cell-SELEX studies present in the literature, it is observed that the total ΔG values of aptamers are variable within a group of negative values [25,26].…”

Encapsulation of hydrophobic components in dendrimersomes and decoration of their surface with proteins and nucleic acids

“…This was exploited to bind DNA oligomers that were conjugated to BG to SNAP-decorated dendrimersomes. An ALC aptamer (EJ4), a DNA therapeutic that has been used for study and treatment of lung carcinomas, was selected for this experiment (50, 51). This 40+ nucleotide ssDNA molecule was also conjugated to a fluorescein amidite (FAM) dye to enable visualization by confocal fluorescence microscopy.…”

Advances in Oligonucleotide Aptamers for NSCLC Targeting

“…A variant of this selection has been then developed by Vidic et al [ 15 ] with the final aim to select aptamers against NSCLC circulating tumor cells with the ability to also recognize cancer-stem like cells. The authors used A549 cells as positive target and human blood cells as negative control.…”

“…More recently, the same authors used two DNA aptamers (ECM-APT-01 and ECM-APT-02) directed against EpCAM to isolate and detect EpCAM + CTCs in clinical blood samples with a detection limit of 0–2 cells/mL of blood [ 20 ]. Further, Vidic and colleagues demonstrated that an aptamer (A155_18) isolated by cell-SELEX (see previous section for details) showed a good potential as a diagnostic tool for the detection of NSCLC CTCs expressing the stem cell marker CD90 [ 15 ].…”