Screening and characterization of high-affinity ssDNA aptamers against anthrax protective antigen.
The protective antigen (PA) of Bacillus anthracis is a secreted protein that functions as a critical virulence factor. Protective antigen has been selected as a biomarker in detecting bacterial infection. The in vitro selection method, systematic evolution of ligands by exponential enrichment (SELEX), was used to find single-stranded DNAs that were tightly bound to PA. After 8 rounds of the SELEX process with PA, 4 different oligonucleotides (referred to as aptamers) that contain a 30-residue ssDNA sequence were identified. Dissociation constant (K(d)) values with Cy3-attached aptamers were determined via fluorophotometry to be within a nanomolar range. The authors attempted to visualize the detection of PA using an aptamer-based enzyme-linked immunosorbent assay method, which has proven to be successful within a nanomolar K(d) value range. Furthermore, 2 of the 4 aptamers exhibited specificity to PA against bovine serum albumin and bovine serum. The results of this study demonstrate the analytical potential of an oligonucleotide-based biosensor for a wide variety of applications, particularly in diagnosing disease through specific protein biomarkers.
Department of Chemistry and Research Institute for Natural Sciences, Hanyang University, Seoul, Republic of Korea.
Ji Sun
JS
Choi
Sang Gon
SG
Kim
Mieke
M
Lahousse
Hye-Yeon
HY
Park
Hae-Chul
HC
Park
Byeongmoon
B
Jeong
Jinheung
J
Kim
Sung-Kun
SK
Kim
Moon-Young
MY
Yoon
D000942:Antigens, Bacterial / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism*; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D015374:Biosensing Techniques; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007700:Kinetics; D009841:Oligonucleotides; D052156:SELEX Aptamer Technique / Q000379:methods
D000942:Antigens, Bacterial; D052157:Aptamers, Nucleotide; D001427:Bacterial Toxins; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; C030325:anthrax toxin