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

Content Provider	Springer Nature Link
Author	Hamula, Camille L. A. Peng, Hanyong Wang, Zhixin Newbigging, Ashley M. Tyrrell, Gregory J. Li, Xing Fang Le, X. Chris
Copyright Year	2015
Abstract	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.
Starting Page	194
Ending Page	209
Page Count	16
File Format	PDF
ISSN	00222844
Journal	Journal of Molecular Evolution
Volume Number	81
Issue Number	5-6
e-ISSN	14321432
Language	English
Publisher	Springer US
Publisher Date	2015-11-04
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Aptamer Cell-SELEX Lactobacillus acidophilus Streptococcus pyogenes Bacteria Evolutionary Biology Microbiology Plant Sciences Plant Genetics & Genomics Animal Genetics and Genomics Cell Biology
Content Type	Text
Resource Type	Article
Subject	Genetics Molecular Biology Ecology, Evolution, Behavior and Systematics

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