The Effects of SELEX Conditions on the Resultant Aptamer Pools in the Selection of Aptamers Binding to Bacterial Cells

Aptamers of high affinity and specificity have a wide range of analytic and clinical applications. Selection of DNA or RNA aptamer molecules usually involves systematic evolution of ligands via exponential enrichment (SELEX), in which a random DNA or RNA library is incubated with a target molecule,...

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Bibliographic Details
Published in:Journal of molecular evolution 2015-12, Vol.81 (5-6), p.194-209
Main Authors: Hamula, Camille L. A., Peng, Hanyong, Wang, Zhixin, Newbigging, Ashley M., Tyrrell, Gregory J., Li, Xing-Fang, Le, X. Chris
Format: Article
Language:English
Subjects:
Animal Genetics and Genomics
Aptamers, Nucleotide - metabolism
Bacteria
Binding sites
Biomedical and Life Sciences
Cell Biology
Cell Wall - metabolism
Cells
Deoxyribonucleic acid
DNA
Evolutionary Biology
Lactobacillus acidophilus
Lactobacillus acidophilus - metabolism
Life Sciences
Ligands
Microbiology
Original Article
Plant Genetics and Genomics
Plant Sciences
Ribonucleic acid
RNA
SELEX Aptamer Technique - methods
Streptococcus pyogenes
Streptococcus pyogenes - metabolism
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Summary:Aptamers of high affinity and specificity have a wide range of analytic and clinical applications. Selection of DNA or RNA aptamer molecules usually involves systematic evolution of ligands via exponential enrichment (SELEX), in which a random DNA or RNA library is incubated with a target molecule, and the oligonucleotides that bind the target are then separated from the nonbinders, PCR amplified, and used as refined libraries in the next round of selection. Conventional SELEX methodologies require the use of purified target molecules and their immobilization onto a solid support. However, purified targets from cells are not always available, and fixing the target to a support may alter its conformation. To overcome these problems, we have developed a SELEX technique using live bacterial cells in suspension as targets, for selecting DNA aptamers specific to cell-surface molecules. Through the selection of aptamers binding to Lactobacillus acidophilus and Streptococcus pyogenes , we report here optimization of this technique and show how varying selection conditions impact the characteristics of resultant aptamer pools, including the binding affinity, selectivity, and the secondary structures. We found that the use of larger starting library sequence diversity, gel purification of the subsequent pools, and the introduction of counter-selection resulted in a more efficient SELEX process and more selective aptamers. A SELEX protocol with lower starting sequence diversity, the use of heat denaturation, and the absence of counter-selection still resulted in high-affinity aptamer sequences specific to the target cell types; however, the SELEX process was inefficient, requiring 20 rounds, and the aptamers were not specific to the strain of the bacterial cells. Strikingly, two different SELEX methodologies yielded the same sequence that bound strongly to the target S. pyogenes cells, suggesting the robustness of the bacterial cell-SELEX technique.
ISSN:0022-2844
1432-1432
DOI:10.1007/s00239-015-9711-y