Selection of aptamers against live bacterial cells.
Single-stranded DNA or RNA aptamer molecules have usually been selected against purified target molecules. To eliminate the need of purifying target molecules on the cell surface, we have developed a selection technique using live bacterial cells in suspension as targets, to select for ssDNA aptamers specific to cell surface molecules. Lactobacillus acidophilus cells were chosen to demonstrate proof of principle based on their high abundance of surface molecules (potential targets). Aptamer pools obtained after 6-8 rounds of selection demonstrated high affinity for and selective binding with L. acidophilus cells when tested via flow cytometry, microscopy, and fluorescence measurements. Out of 27 aptamers that were cloned and sequenced, one sequence, hemag1P, was found to bind to L. acidophilus much more strongly and specifically than other cells tested. This aptamer was predicted to have a tight hairpin secondary structure. On average, an estimated 164 +/- 47 aptamer molecules were bound to a target cell with an apparent K d of 13 +/- 3 nM. A likely putative molecular target of hemag1P is the S-layer protein on the cell surface.
Hamula
Camille L A
CL
Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, 10-102 Clinical Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2G3.
Zhang
Hongquan
H
Guan
Le Luo
LL
Li
Xing-Fang
XF
Le
X Chris
XC
D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism*; D001419:Bacteria / Q000166:cytology* / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D015723:Gene Library; D007779:Lactobacillus acidophilus / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D019936:Probiotics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D052157:Aptamers, Nucleotide