| Autor: |
Hamula CL; Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, 10-102 Clinical Sciences Building, Edmonton, AB, T6G 2G3, Canada.; Mount Sinai Hospital, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, New York City, NY, 10029, USA., Peng H; Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, 10-102 Clinical Sciences Building, Edmonton, AB, T6G 2G3, Canada., Wang Z; Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, 10-102 Clinical Sciences Building, Edmonton, AB, T6G 2G3, Canada., Newbigging AM; Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, 10-102 Clinical Sciences Building, Edmonton, AB, T6G 2G3, Canada., Tyrrell GJ; The Provincial Laboratory for Public Health for Alberta, Walter Mackenzie Health Sciences Centre, Edmonton, AB, T6G 2J2, Canada.; Department of Laboratory Medicine and Pathology, 2B3.08 Walter Mackenzie Health Sciences Centre, University of Alberta, Edmonton, AB, T6G 2B7, Canada., Li XF; Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, 10-102 Clinical Sciences Building, Edmonton, AB, T6G 2G3, Canada. xingfang.li@ualberta.ca., Le XC; Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, 10-102 Clinical Sciences Building, Edmonton, AB, T6G 2G3, Canada. xc.le@ualberta.ca. |
| Abstrakt: |
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. |
