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

Mateja Vidic
Mateja Vidic
, 
Tina Smuc
Tina Smuc
, 
Nika Janez
Nika Janez
, 
Michael Blank
Michael Blank
, 
Tomaz Accetto
Tomaz Accetto
, 
Jan Mavri
Jan Mavri
, 
Isis C. Nascimento
Isis C. Nascimento
, 
Arthur A. Nery
Arthur A. Nery
, 
Henning Ulrich
Henning Ulrich
 and 
Tamara T. Lah
Tamara T. Lah
   | Mar 25, 2018
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Article Category: Research Article
Published Online: Mar 25, 2018
Page range: 152 - 159
Received: Nov 24, 2017
Accepted: Feb 26, 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.

Figure 1

Flow cytometry of cells double labelled with CD90 and aptamers. (A) Flow cytometry was performed for FITC-labelled aptamer pools comparing binding to A549 cell binding of the pool of the library), the sixth cycle and the pool following negative selection cycle against blood cells. The density plots are showing that there was no difference in the percentage of labelled cells for each of the pools. Displacement of the population in the density plot, and shift of position on the histograms for the cells labelled with the pool, which followed the negative selection against blood cells, was observed, showing a cell population with increased fluorescence intensity compared to the cells labelled with the initial library or pool of 6th cycle. (B) CD90 binding and cell sorting: After adjustment of cellular gates for un-labelled cells, CD90+, the library and the pool following negative selection, the double-labelled cells labelled for CD90/library and CD90/negative selection pool were compared. Here, the gate was set to collect the most intensely labelled cells with the aptamer pool following negative selection. To confirm the profile of the isolated population, the sorted cells were analysed again by flow cytometry (post sort). The density plots show the presence of a well-defined secondary population that was double labelled, suggesting that aptamers were recognizing a sub-population of CD90, which was positive for stem cells within the A549 cell population.
Flow cytometry of cells double labelled with CD90 and aptamers. (A) Flow cytometry was performed for FITC-labelled aptamer pools comparing binding to A549 cell binding of the pool of the library), the sixth cycle and the pool following negative selection cycle against blood cells. The density plots are showing that there was no difference in the percentage of labelled cells for each of the pools. Displacement of the population in the density plot, and shift of position on the histograms for the cells labelled with the pool, which followed the negative selection against blood cells, was observed, showing a cell population with increased fluorescence intensity compared to the cells labelled with the initial library or pool of 6th cycle. (B) CD90 binding and cell sorting: After adjustment of cellular gates for un-labelled cells, CD90+, the library and the pool following negative selection, the double-labelled cells labelled for CD90/library and CD90/negative selection pool were compared. Here, the gate was set to collect the most intensely labelled cells with the aptamer pool following negative selection. To confirm the profile of the isolated population, the sorted cells were analysed again by flow cytometry (post sort). The density plots show the presence of a well-defined secondary population that was double labelled, suggesting that aptamers were recognizing a sub-population of CD90, which was positive for stem cells within the A549 cell population.

Figure 2

Predicted secondary structures of four aptamer candidates selected for in vitro experiments. The secondary structures of sequences were enabling formation of complementary wrapping around their target. The unpaired bases were responsible for binding events where Watson-Crick paired bases were giving the aptamer stability. Predicted secondary structures were calculated with UnaFold, the presented are the ones with lowest ΔG, i.e. the highest stability (energy rules: DNA, temperature 24°C, 14.4 mM NaCl, 3.2 mM MgCl). Equilibrium dissociation constants Kd (nM) were calculated using GraphPad Prism 5, under the non-linear fit model, one-site non-competitive binding to fluorescent population ratio at used aptamer concentrations. See the Supplementary Materials for further details.
Predicted secondary structures of four aptamer candidates selected for in vitro experiments. The secondary structures of sequences were enabling formation of complementary wrapping around their target. The unpaired bases were responsible for binding events where Watson-Crick paired bases were giving the aptamer stability. Predicted secondary structures were calculated with UnaFold, the presented are the ones with lowest ΔG, i.e. the highest stability (energy rules: DNA, temperature 24°C, 14.4 mM NaCl, 3.2 mM MgCl). Equilibrium dissociation constants Kd (nM) were calculated using GraphPad Prism 5, under the non-linear fit model, one-site non-competitive binding to fluorescent population ratio at used aptamer concentrations. See the Supplementary Materials for further details.
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